Chapter 7
Impact of Harsh Environmental Conditions on Nodule
Formation and Dinitrogen Fixation of Legumes
Mohamed Hemida Abd-Alla, Ahmed A. Issa and
Takuji Ohyama
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/56997
1. Introduction
Dinitrogen (N2) comprises almost 80% of the atmosphere. The triple-bonded molecule
cannot be used directly by higher plant. The element nitrogen, or “azote,” meaning “with‐
out life,” as Antonie Lavoisier called it about 200 years ago, has proved to be anything
but lifeless, since it is a component of food, poisons, fertilizers, and explosives (Schoot Ui‐
terkamp 1990). The atmosphere contains about 1015 tonnes of N2 gas, and the nitrogen cy‐
cle involves the transformation of some 3 × 109 tonnes of N2 per year on a global basis
(Postgate 1982). However, transformations (e.g., N2 fixation) are not exclusively biologi‐
cal. Lightening probably accounts for about 10% of the world’s supply of fixed nitrogen
(Sprent and Sprent 1990). The fertilizer industry also provides very important quantities
of chemically fixed nitrogen. World production of fixed nitrogen from dinitrogen for
chemical fertilizer accounts for about 25% of the Earth’s newly fixed N2, and biological
processes account for about 60%. Nitrogen availability is probably the second most limit‐
ing factor in agricultural production, second only to water availability (Date 2000). The le‐
gume-Rhizobium symbiosis is the single most important source of biologically fixed
nitrogen in agricultural systems (Graham and Vance 2000). Since it is a biological proc‐
ess, it does not depend on external sources of energy, except for free and renewable sun‐
light, and has few detrimental ecological effects (Phillips 1999).
2. Importance of biological nitrogen fixation by legumes
The legume-Rhizobium symbiosis allows many species to obtain their nitrogen nutrition
from N2. This has been known since the Greeks discovered the value of legumes in enhanc‐
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ing soil fertility, some 2300 years ago. Since 1888 it has been known that this is due to the
nodules on legume roots and the bacteria in them (Date 2000). This nitrogen fixation is very
hard to quantify in economic (Lanyon 1995) or ecological terms (Hoglund and Brock 1987),
but is estimated to be responsible for between 45% (Danso 1995) and 85% (Vance et al. 1988)
of the biological nitrogen fixation in agricultural areas. Biological nitrogen fixation (BNF)
has the advantage of being environmental friendly and therefore would be ideal for sustain‐
able agriculture. Biological nitrogen fixation, a key source of N for farmers using little or no
fertilizer, constitutes one of the potential solutions and plays a key role in sustainable grain
legumes production. Given the high cost of fertilizer in developing countries and the limited
market infrastructure for farm inputs, current research and extension efforts have been di‐
rected to integrated nutrient management, in which legumes play a crucial role (Chianu et
al. 2008). Certain microorganisms have the ability to use the renewable source of energy to
fix atmospheric nitrogen under mild conditions, such as normal temperature and normal
pressure. Nitrogen fixation is a key process in which molecular nitrogen is reduced to form
ammonia, which is the form of nitrogen that is used by living systems for the synthesis of
many bioorganic compounds. Biologically-fixed nitrogen could be directly “absorbed” by
plants and keep the environment almost “untouched”. Crop rotation with legumes has been
recognized to increase soil fertility and agricultural productivity since ancient China and
Rome (Cheng 2008). Rhizobia live in the rhizosphere of leguminous plants such as soybeans
and faba bean, forming colonies that produce root nodules (Foth 1990). Biological nitrogen
fixation is an oxygen dependent reaction and therefore is prevalent in legumes growing in
aerated, upland soils. Currently, approximately 2 tons of industrially-fixed nitrogen are
needed as fertilizer for crop production to equal the effects of 1 ton of nitrogen biologicallyfixed by legume crops. Therefore, biologically-fixed nitrogen influences the global nitrogen
cycle substantially less than industrially-fixed nitrogen. One day, this situation needs to be
changed. Research in this field has pivotal importance and would be significantly beneficial.
Inoculation with compatible and appropriate rhizobia may be necessary where a low popu‐
lation of native rhizobial strains predominates and is one of the solutions which grain le‐
gume farmers can use to optimize yields. It is critical for sustained yield in farmlands
deficient in native rhizobia and where N supply limits production. Research on use of Rhi‐
zobium inoculants for production of grain legumes showed it is a cheaper and usually more
effective agronomic practice for ensuring adequate N nutrition of legumes, compared with
the application of N fertilizer (Chianu et al 2008). The major findings are: (1) complete ab‐
sence of or very weak institutions, policy and budgetary support for biotechnology research
and lack of its integration into wider agricultural and overall development objectives, (2)
limited knowledge of inoculation responses of both promiscuous and specifically nodulat‐
ing legume varieties as well as the other factors that inhibit BNF, hence a weak basis for de‐
cision-making on biotechnology issues, (3) limited capacity and lack of sustainable
investment, (4) poorly developed marketing channels and infrastructure, and limited in‐
volvement of the private sector in the distribution of inoculants, and (5) limited farmer
awareness about and access to (much more than price) inoculants. The lessons learned in‐
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clude the need: (1) to increase investment in Rhizobium inoculation technology development,
and strengthen policy and institutional support, (2) for public private partnership in the de‐
velopment, deployment and dissemination of BNF technologies, (3) to develop effective
BNF dissemination strategies (including participatory approach) to reach farmers, and (4)
for greater emphasis on capacity building along the BNF value chain. According to an FAO
report, production of N fertilizer for 2007 was 130 million tons of N, and this should further
increase in the coming years (FAO 2008). This extensive use has certain drawbacks. A pro‐
portion of added fertilizer is lost as a result of denitrification and leaching of soil by rainfall
and irrigation. In addition, leaching leads to water pollution is caused by eutrophication. As
a consequence, extending application of biological nitrogen fixation by any means is an im‐
portant issue.
The legume-Rhizobium symbiosis is the single most important source of biologically fixed ni‐
trogen in agricultural systems (Graham and Vance 2000); although under some conditions
free-living and associative systems are important (Phillips 1999). Since it is a biological proc‐
ess, it does not depend on external sources of energy, except for free and renewable sun‐
light, and has few detrimental ecological effects (Phillips 1999). Conversely, chemical
nitrogen fixation depends on fossil fuels (generally natural gas), both as an energy source
and as a reductant source (Braswell et al. 1997). In addition, extensive use of nitrogen based
fertilizers tends to be inefficient, with absorption by the crop ranging from 20 to 50 % of ap‐
plied nitrogen (Graham and Vance 2000). Much of the nitrogen not absorbed by crops can
find its way into ground water, lakes and rivers, where it poses both health and environ‐
mental problems (Graham and Vance 2000). Since the legume-bacteria symbiosis is so im‐
portant in agriculture, even a slight increase in its efficiency, in terms of total nitrogen
fixation, would have a major impact a global nitrogen inputs, and increase the efficiency of
production for the corresponding cropping system (Phillips 1999). The interaction between
legumes (Leguminosae) and bacteria in the family Rhizobiaceae leads to the development of
a nitrogen-fixing symbiosis (Ohyama et al. 2009). Several environmental factors can adverse‐
ly affect the performance of symbiotic nitrogen fixation by legumes. These factors can inter‐
vene at the following levels: survival of rhizobia in the soil, the infection process, nodule
growth and nodule function. Also, these factors can affect N2 fixing performance indirectly
through their effects on host plant growth.
3. Effect of harsh environmental conditions on survival of rhizobia in
agroecosystems and process of nodulation and nitrogen fixation of
legumes
Rhizobia is the common name given to a group of small, rod-shaped, Gram-negative bacte‐
ria that collectively have the ability to produce nodules on the roots of leguminous plants
and belong to the family Rhizobiaceae, which are part of the α-proteobacteria. In early stud‐
ies, the taxonomy of rhizobia was based on the rate of growth of isolates on laboratory me‐
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dia and their selective interaction with their plant hosts. It was soon established that no
strain could nodulate all plants, but that each could nodulate some legumes though not oth‐
ers (Long 1989). This led to the concept of cross-inoculation groups, with organisms group‐
ed according to the hosts they nodulated. Within the genus Rhizobium several strains
nodulate a common host, but are distinct according to genetic and/or phenotypic properties
and are therefore classified as distinct species (e.g. R. tropici and R. etli). For a time this was
the basis on which rhizobia were identified. However, developments in molecular biology
and advances in bacterial taxonomy (Graham et al. 1991) have resulted in a rhizobial taxono‐
my based on a wide range of characteristics and to the distinction of new genera and spe‐
cies. Rhizobia were confined to the Alphaproteobacteria, namely in the order Rhizobiales.
The Alphaproteobacteria class comprises 11 genera that include bacteria able to induce nod‐
ule formation in legume plants: Agrobacterium, Allorhizobium, Azorhizobium, Bradyrhizobium,
Devosia, Mesorhizobium, Methylobacterium, Ochrobactrum, Phyllobacterium, Rhizobium and Ensi‐
fer (Euzéby 1997). Agrobacterium genus has been proposed by Young et al. (2001) to be
emended and reclassified as Rhizobium. The members of the genus Sinorhizobium were trans‐
ferred to the genus Ensifer by decision of the Judicial Commission of the International Com‐
mittee on Systematics of Prokaryotes (Tindall 2008). Three additional genera belonging to
the Betaproteobacteria class were recently added to the list of genera containing rhizobia spe‐
cies, namely, Burkholderia (Moulin et al. 2001), Cupriavidus (Chen et al. 2001,2003) and Her‐
baspirillum (Valverde et al. 2003). It is noteworthy to mention that these 14 genera also
include a variety of non-symbiotic bacteria. More recently a member of the γ-proteobacteria
has been found that also nodulates legumes (Benhizia et al. 2004).
When studying any living organism, it is important to know how each species grows and
responds to certain conditions that can be found in their natural environment. Ascertaining
how bacteria respond to environmental signals, or stressful conditions, is a vital part to un‐
derstanding how those microbes live, thrive and survive. Every bacterium has optimum
conditions that make this process easier, however in order to survive in a changing environ‐
ment (or some other form of stress) the bacteria must be able to adapt. This adaptation is a
stress response. Two types of stress responses operate in microorganisms: the general stress
response and specific stress responses. The general stress response is normally controlled by
a single or a few master regulators (Bremer and Krämer 2000) and provides cross-protection
against a wide variety of environmental cues, regardless of the initial stimulant (Hecker et
al. 1996; Hecker and Völker 1998). This response is effective in allowing the cell to survive,
but it may not be enough to let the cell grow under the stressful conditions (Bremer and
Krämer 2000). Under prolonged stress conditions cells employ specific stress responses,
which utilize highly integrated networks of genetic and physiological adaptation mecha‐
nisms (Bremer and Krämer 2000). Usually, there is also a complex relationship between cel‐
lular response systems and global regulators, adding another level of control to the cell’s
emergency stress response and long-term survival reactions (Hengge-Aronis 1999). Al‐
though the above description is usually what happens, not all general responses occur im‐
mediately on stressful stimuli as some activate on entry into stationary phase; likewise some
specific stress response are induced as soon as stress is detected.
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3.1. Temperature
3.1.1. Survival of Rhizobia
Soil environmental conditions are critical factors to the persistence and survival of rhizobia
in the soil. The changes in the rhizospheric environment can affect both growth and sapro‐
phytic competence, which will influence competitiveness and persistence (Dowling and
Broughton 1986). In arid and semiarid regions of the tropics, the soil temperatures near the
surface can be very high. In Egyptian sandy soils, the temperature near the soil surface was
59 °C at the air temperature 39°C. However, the soil temperature decreased rapidly with
depth, being moderate 35 °C, at 15 cm. It appears, however, that rhizobia are more resistant
to high temperatures in soil than in laboratory medium (AbdelGadir and Alexander 1997).
The study performed with Indian desert soils suggested that not the high soil temperature
but the low organic matter and poor soil moisture were the major factors that reduced the
numbers of different micro-organisms (Rao and Venkateswarlu 1983). Indeed, in droughtaffected Acacia senegal soils the numbers of culturable rhizobia were significantly reduced,
approximately from 107 to 106 cfu g-1. In conclusion, one can assume that during the dry sea‐
son, the water deficit together with the high soil temperature will considerably decrease rhi‐
zobial numbers or cause a lack of rhizobia in the surface soils (0-10 cm). In order to improve
the yield of legumes in more adverse environments, stress-tolerant cultivars should be com‐
bined with stress tolerant rhizobia. Studies on rhizobia biodiversity are an important ap‐
proach to find more tolerant strains, even when non-adverse environments are sampled,
since populations often contain tolerant strains to non-acting stresses, as resilience to re‐
spond to future problems (Giller et al. 1997). The ability of rhizobia to persist in the absence
of their host plant is perhaps more dependent of their ability to endure adverse environmen‐
tal factors than during symbiosis, where the nodule represents a protective environment.
Environmental stresses especially during the period between inoculation of seeds and ger‐
mination impose severe problems to establishment of a successful symbiosis (Weaver and
Holt 1990; Weaver et al. 1985). Every bacterium has its own optimum conditions, under
which it grows at its best. For most rhizobia, the optimum temperature range for growth is
28 – 31 °C, and many are unable to grow at 37°C (Zahran 1999). Not only do the bacteria
themselves have an optimum temperature range, but the processes within them do as well.
Survival of Rhizobium leguminosarum, the microsymbiont of faba bean, may be affected main‐
ly by extremes in temperature. Differences in adaptation to high temperatures have been
demonstrated from different climatic zones. Eaglesham and Ayanaba (1984) reported that
more than 90 % of cowpea rhizobia isolated from hot dry Sahelian Savanna in Niger were
able to grow at 40 °C while the rhizobia isolated form cooler humid regions of West Africa
did not grow at this temperature (Bowen and Kennedy 1956; Munevar and Wollum 1981).
Some rhizobial strains isolated from nodules in arid environments are able to grow at 40 °C
or even higher (Eaglesham et al. 1981). Kluson et al. (1986) reported differences in the toler‐
ance of bradyrhizobia cultivated in liquid medium and in soil to temperatures within the
range of 20 to 35 °C. Gewaily et al. (1991) similarly reported that survival of Rhizobium legu‐
minosarum was significantly reduced at 40 °C when inoculated in sterile and non sterile soils.
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Baldani and Weaver (1992) attributed heat tolerance of Rhizobium leguminosarum biovar trifo‐
lii strains to cryptic plasmids. These plasmids induce the synthesis of heat shock proteins
upon exposure of bacteria above normal growth temperatures (Sen et al. 1990). Abd-Alla
and Abdel-Wahab (1995a) reported that Strain RCR 1001 was more resistant to heat and
nodulated faba bean better than other tested strains. Although strain adaptation to high
temperature has been reported, it has been associated with decreased effectiveness in estab‐
lishing symbiosis, mostly due to plasmid loss (Hungria and Franco 1993). Additionally, heat
stress has been shown to induce the synthesis of heat shock proteins in Rhizobium (Michiels
et al. 1994) and to modify lipopolysaccharide patterns and bacterial mobility (Zahran et al.
1994). Studies on rhizobia biodiversity are an important approach to find more tolerant
strains, even when non-adverse environments are sampled, since populations often contain
tolerant strains to non-acting stresses, as resilience to respond to future problems (Giller et
al. 1997). Temperature is often pointed out as the major factors in determining the bacterial
community diversity (Fierer and Jackson 2006; Staddon et al. 1998).
The optimum temperature for rhizobia growth is 25-30 °C (Zhang et al. 1995), however in
both saprophytic and symbiotic life rhizobia are often subject to temperatures out of this
range. Most studies in rhizobial temperature stress tolerance focus soybean and commonbean microsymbionts. Soybean isolates grow weakly at 40 °C and no isolate was able to
grow at 45°C (Chen et al. 2002). Rhizobia nodulating P. vulgaris can survive to 47 °C, but
their symbiotic effectiveness is loss at high temperatures; while other isolates tolerant to 40
°C were able to remain infective at that temperature (Karanja and Wood 1988). Nandal et al.
(2005) reported that mutants tolerant to high temperature (43 °C), obtained from a thermo‐
sensitive Rhizobium sp. strain, exhibited a different protein profile from that of the wild type
at high temperature, namely the mutant strains showed overexpressed proteins and new
proteins. A protein of 63-75 kDa was overproduced in all mutant strains, which probably
corresponds to DnaK. In chickpea rhizobia, a 60 kDa protein that could correspond to
GroEL was found to be consistently overproduced when isolates were submitted to heat
stress (Rodrigues et al. 2006). Strains isolated from a chickpea wild relative (Cicer anatoli‐
cum), collected from high altitudes, were successful in nodulating chickpea at low tempera‐
tures, what represents an alternative source of chickpea nodulating rhizobia with potential
use as inoculants (Ogutcu et al. 2008). The changes in the population occupancy of some bra‐
dyrhizobia tested were in accordance with the geographical distribution of indigenous soy‐
bean-nodulating bradyrhizobia (Saeki et al. 2006). Saeki et al. (2010) reported that the
occupancies of bradyrhizobia in soil microcosms after long-term incubation may change de‐
pending on the incubation temperature. They suggest that soil temperature may be one im‐
portant environmental gradient determining bradyrhizobial niches from northern to
southern regions of Japan. Temperature stress is generally divided into two classes: heat
shock and cold shock. Bacterial heat shock is the more characterised of the two (Phadtare et
al. 2000). The heat shock response is very similar to the acid stress response, in that many
proteins with a similar mode of action are synthesised. Heat shock proteins contribute to
heat tolerance by conferring heat protection on the bacteria but do not alter the internal tem‐
perature of the cell (Yura et al. 2000). Like acid shock protein, there are two main types of
heat shock protein: chaperones and proteases. These work in the same way as the cid shock
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proteins. Heat shock proteins, and their regulation, structure and function, have been stud‐
ied in great detail. Their function appears to be highly conserved between both prokaryotes
and eukaryotes (Netzer and Hartl 1998). Some of these proteins are also vital under normal
(non-heat shock) growth conditions (Münchbach et al. 1999; Lentz 2004).
Cold shock is essentially the opposite of heat shock. Instead of proteins misfolding and de‐
naturing, cells undergoing cold shock have to contend with a loss of membrane and cytosol
fluidity and with the stabilisation of secondary structures of RNA/DNA (Phadtare et al.
2000). RNA/DNA stabilisation leads to a decrease in the efficiency of translation, transcrip‐
tion and replication. Bacterial cold shock response is an immediate and transient response to
the temperature downshift. This is followed by low temperature adaptation that allows con‐
tinued growth at low temperatures (Panoff et al. 1997). Generally, bacteria overcome loss of
fluidity by increasing the amount of unsaturated fatty acids in the membrane phospholipids
(Phadtare et al. 2000). Cold shock response also leads to the production of many cold shock
proteins (CSPs). Just like ASPs and HSPS, these too are mainly chaperones and proteases
(Phadtare et al. 2000). However, instead of protecting against the misfolding of proteins, the
CSP chaperones are primarily used to bind to RNA/DNA to prevent stabilisation and allow
translation and transcription to proceed as usual (Phadtare et al. 2000). CspA is an RNA
chaperone and a major CSP found in many bacteria (Jiang et al. 1997). A CspA homologue is
present in Sinorhizobium meliloti and is induced following a temperature downshift from 30
to 15 °C, along with the three rRNA (rrn) operons. It is unknown what function the genes
and products of the rrn operons or CspA have in response to cold shock, as mutations made
in these genes showed no change in cell phenotype at 15 °C compared to the wild-type
(O’Connell et al. 2000; Gustafson et al. 2002). TypA is also required for growth at low tem‐
peratures and is believed to act as a regulator by controlling the phosphorylation of proteins
(Kiss et al. 2004). Expression of several heat shock operons, mainly coding for small heat
shock proteins, is under the control of repression of heat shock gene expression in various
rhizobial species (Nocker et al. 2001). The molecular bases of temperature stress tolerance in
rhizobia were investigated, by comparing the expression of chaperone genes dnaKJ and
groESL in thermotolerant and thermosensitive isolates. Tolerance to cold, heat and heat
shock was evaluated for 53 mesorhizobia (Alexandre and Oliveira 2011). The analysis of the
dnaK and groESL expression by Northern hybridization, using isolates from three species
groups, showed an increase in the transcripts levels with heat, but not with cold stress. Lar‐
anjo and Oliveira (2011) reported that under heat stress a protein of approximately 62 kDa
was newly detected in Mesorhizobium chacoense. The over expression of a 62 kDa protein in
Mesorhizobium huakuii and Mesorhizobium septentrionale was controlled groEL expression and
increased upon heat shock. Interestingly, a 62 kDa protein, the NdvCprotein, has been de‐
scribed as important in osmoregulation, associated with symbiotic systems in B. japonicum
(Bhagwat et al. 1996). Further studies are required to elucidate the function of overexpressed
genes in order to clarify their role in environmental stress tolerance of rhizobia, as well as
their contribution to symbiotic effectiveness (Alexandre and Oliveira 2012).
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3.1.2. Rhizobium- Legume molecular signalling exchange
Molecular signalling in the rhizosphere controls the nature of relationships between plants
and other soil organisms. For instance, legume-derived signals, such as betaines and isofla‐
vonoids, function by chemoattracting rhizobia and trigger the events that lead to endosym‐
biosis. The molecular signalling exchange between both microsymbiont and legume is
fundamental for an effective legume-rhizobium symbiosis and can determine the specificity
of this symbiotic relationship. Production of Nod factors or lipo-chito-oligosaccharide sig‐
nalling molecules by the prokaryotic partner is activated by the release of plant phenolic sig‐
nals, mainly flavonoids, into the rhizosphere. The phenolic flavonoid compounds partly
determine the specificity of the symbiotic relationship as each Rhizobium species responds to
specific flavonoids. Another determinant of host symbiont specificity is attributed to the dif‐
ferent Nod factors substituents attached to the oligosaccharide backbone (Dénarié et al.
1996; Oldroyd 2001). Production of Nod factors or lipo-chito-oligosaccharide signalling mol‐
ecules by the prokaryotic partner is activated by the release of plant phenolic signals, mainly
flavonoids, into the rhizosphere. The phenolic flavonoid compounds partly determine the
specificity of the symbiotic relationship as each Rhizobium species responds to specific flavo‐
noids. Another determinant of hos symbiont specificity is attributed to the different Nod
factors substituents attached to the oligosaccharide backbone (Dénarié et al. 1996; Oldroyd
2001). Most rhizobia species interact with only a few select legumes, but some have been
shown to have a broad host range (Pueppke and Broughton 1999). For example, the strain
Ensifer sp. NGR234 is able to nodulate over 120 plant genera, including the non-legume Par‐
asponia andersonii. This feature may depend up on the family of Nod factors secreted, which
are more diverse than in all other rhizobia known (Schmeisser et al. 2009) and in the concen‐
tration of Nod factors released by the NGR234 that is much higher than usual. More recent‐
ly, M. opportunistum WSM2075 was isolated from Biserrula pelecinus root nodules, but the
symbiotic genes of this organism provide a broader range of hosts for nodulation, including
also Astragalus adsurgens, A. membranaceus, Lotus peregrinus and Macroptilium atropurpureum
(Nandasena et al. 2009). On the other hand, there are legumes species that can be nodulated
by several rhizobia species and others that are very restrict for nodulation and only accept
as microsymbionts a reduced number of species. For example, Phaseolus vulgaris is known as
a promiscuous host, since it can be nodulated by rhizobia belonging to diverse genera (such
as Bradyrhizobium, Rhizobium and Ensifer) while Cicer arietinum is considered a restrict host,
because it is nodulated only by Mesorhizobium species. Nevertheless, the host range depends
on the legume cultivar used and conditions tested (Martinez-Romero 2003).
Temeprature play a critical role on the exchange of molecular signals between rhizobia and
their host, thus reducing nodulation. Low temperature inhibits inter-organismal signaling
between the two symbiotic partners. It has been shown that low temperature inhibits the bi‐
osynthesis and rhizosecretion of plant to-bacteria signal molecules (for example genistein
from soybean roots, which are necessary for the induction of the nod genes of B. japonicum
(Zhang and Smith 1997; Abd-Alla 2001, 2011). Low temperature also inhibit the induction of
bacterial nodulation genes (nod gene) required for the biosynthesis of bacteriato-plant sig‐
naling molecules, lipo-chitooligosaccarides (LCOs), the so-called Nod factors (Zhang et al.
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1995). The disruption of Nod factor production/excretion at low temperature incubations
has been previously reported in R. leguminosarum bv. trifolii (McKay and Djordjevic 1993),
and in B. japonicum (Duzan et al. 2005). Low temperatures delay the onset of nodulation
(Pan and Smith 1998) and reduce the rate of subsequent nodule growth, resulting in effects
on small final nodule size (de Lira Juniora et al. 2005). The presence of appropriates flavo‐
noids in root exudates is a critical factor in nodule formation (Richardson et al. 1988 a,b) and
dictate rhizobia legume specificity. The nodulation status of the pea (Pisum sativum L.) was
most profoundly improved via the addition of the flavonoid naringenin (Bandyopadhyay et
al. 1996), possibly via the induction of nod gene expression in R. leguminosarum. In the soy‐
bean-B. japonicum symbiosis, Kosslak et al. (1987) reported that genistein was the most effec‐
tive inducer for the expression of the nodYABC operon of B. japonicum. Exogenous
application of genistein results in the short circuiting of plant-bacterium signaling, and has
been confirmed as an effective means to mitigate the adverse effects of low temperature on
nodulation and nitrogen fixation (Zhang and Smith 1997). Pre-incubation of B. japonicum
with genistein hastened the onset of nitrogen fixation, and increased the number and size of
the nodules and plant growth. Additionally, this beneficial effect of genistein increased with
decreasing temperature (Zhang et al. 1995). Firmin et al. (1993) and Ovtsyana et al. (1999)
observed that nodule formation on cv. Afghanistan inoculated with R. leguminosarum bv.
viciae is at least partly controlled by Nod-factors that carry a modification on the reducing
terminus (an acetyl group encoded by the acetyl transferase NodX ) as well as by a single
genetic locus sym2A in the host-plant. R. leguminosarum bv. viciae strains producing Nod
factors lacking this modification cannot nodulate these pea plants at low temperatures.
Somehow, blockage of nodulation is overcome at higher temperatures suggesting a temper‐
ature-sensitive gene-for-gene relationship between nodX and sym2A (Kozik et al. 1995; Lie
1984; Olsthoorn et al. 2000). There is, however, little information available about the effect of
high temperature on Rhizobium- Legume molecular signalling exchange, additional studies
are required.
3.1.3. Rhizobia-legume nodulation and nitrogen fixation
The interaction between Rhizobium and host legume results in the formation of root nodules.
The relation between rhizobia and legume is a selective one: individual species of rhizobia
have a distinct host range, from narrow to broad, allowing nodulation of a particular set of
leguminous species. Also, a particular leguminous species can be infected by a certain range
of rhizobia. Development of functional nodules requires temporally and spatially controlled
activity of genes and gene products of both partners (Oldroyd et al. 2001). The Rhizobiumlegume interactions are controlled by signal exchange between the two partners. Legumes
secrete specific flavonoid-type molecules (Gagnon and Ibrahim 1998). These compounds are
main components in the chemical communication between symbiotic legumes and different
species of nitrogen-fixing bacteria leading to nodule formation and N2 fixation (Werner
2001). Flavonoid perception attracts the specific rhizoba to the root hairs and activates nodu‐
lation (nod) gene expression, via the bacteria activator NodD (Lindström et al. 2002). NodD
activates transcription of nod boxes promoters, and represents the first level of host-specific
recognition (Schultze and Kondorosi 2008). The nod genes expression lead to the production
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of strain-specific lipo-chito-oligosaccharides, also called as Nod factors (Spaink 2000). Nod
factors are considered the second level of host-specific recognition (Perret et al. 2000). Nodfactors induce various plant responses, including root-hair deformation, cortical cell-divi‐
sion, ‘pseudo-nodule’ and nodule-formation (Broughton et al. 2003). On production of Nod
factors, the rhizobia then surround and attach to the tip of root, causing the root to start to
curl (Yao and Vincent 1969). Rhizobia trapped in a curled hair, or between a hair and anoth‐
er cell, proliferate and begin to infect the outer plant cells, which in turn stimulates plant
cells to produce infection threads (Callaham and Torrey 1981). Rhizobia are released from
infection threads into the cytoplasm of plant cells. The Rhizobia are surrounded by plasma
membrane of plant origin and then briefly replicate their DNA and divide before stopping
both processes (Robertson et al. 1978). The membrane-enveloped rhizobia continue to divide
within the host cells before they differentiate into bacteroids and start to fix nitrogen (Roth
and Stacey 1989a, b). The endosymbiotic forms of the rhizobia are referred to as bacteroids
and begin to fix nitrogen by the action of the enzyme nitrogenase (Xi et al. 2000). Atmos‐
pheric nitrogen is converted into ammonia by bacteroids and is subsequently assimilated in‐
to the plant following its conversion to glutamine by glutamine synthase. Within the nodule
interior and the neighboring plant cells, essential nutrients are exchange between bacteroids
and plant cells through a symplastic and apoplastic pathway (Abd-Alla et al. 2000 a).
Temperature regulates the metabolism of the plant and the bacteria, as well as the plant–
bacteria association (Young et al. 2006). Sometimes, the sensitivity of the host toward low
temperature affects nitrogen fixation severely, leading to an abrupt cut-off at tempera‐
tures where the bacterial cells can still grow and metabolize. Root hair infection is much
more temperature sensitive than nodule development (Hafeez et al. 2000). Naeem et al.
(2008) suggested that suboptimum temperatures affect the growth of bacteroids even
within the nodule and also affect the cell-to-cell movement of the bacteroids. Most of the
work related to low soil temperature effects on signal exchange and nodulation has been
conducted by Smith group on soybean. The root zone ternperatures blow 17.5 °C cause
reduction and delays nodule formation and the onset of nitrogen fixation (Zhang and
Smith 1996). Soil temperatures below 25 °C are prevalent in Canada until mid-June/midJuly (Zhang and Smith 1994). This delay reduces the time during which there is nitrogen
fixation by more than 10 days (Zhang and Smith 1995). This leads to decline in total nitro‐
gen fixation and lower crop yield (Lynch and Smith 1994), and can be at least partially
corrected by the addition of genistein (Zhang and Smith 1997; Pan and Smith 1998). It is
known, however, that low soil temperatures affect nodule initiation (Cordovilla et al 1999)
and function (Rice et al. 1995).A greater concen in the tropical zone, but also important
during the peak of summer in temperate zones., is excessive soil temperature and its abil‐
ity to be an important limitation to nodulation and nitrogen fixation (Hungria and franco
1993; Boddey et al. 1997; Graham and Vance 2000). Hungria and Vargas (2000) note that
maximum soil ternperature in the tropics regularly exceed 40 °C at 5 cm below the soil
surface and 50 °C at 1 cm. Such high temperatures can limit nodulation. The range for op‐
timal growth of legurnes dependent on nitrogen fixation have been recognized to be nar‐
rower than for nitrogen-fed legumes since at least (Hungria and Vargas 2000). Elevated
temperature inhibited plant growth by diminishing net photosynthesis and nitrogen fixa‐
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tion (Aranjuelo et al. 2007). The optimum temperature for nodule formation is ranged be‐
tween 25 and 33 °C (Pankhurst and Sprent 1976), although decline in nitrogenase activity
have been recorded above 28 °C (Hungria and Franco 1993). High temperature might af‐
fect N2 fixation directly or indirectly. Direct inhibition by temperature is a consequence of
decreased nodule development (Dart and Mercer 1965; Piha and Munns 1987), activity
(Meyer and Anderson 1959; Piha and Munns 1987; Hernandez-Armenta et al. 1989) and
accelerated nodule aging. Indirect inhibition is related to temperature effects on root hair
formation depression, decrease of nodulation sites (Frings 1976; Jones and Tisdale 1921),
and modified adherence of bacteria to root hairs (Frings 1976). Moreover, Aranjuelo et al.
(2007) observed that high temperature negatively affected bacteroid enzyme activity re‐
gardless of water availability, although the inhibitory effect on the bacteroid fraction was
even greater. The decrease in malate dehydrogenase activity was observed in plants ex‐
posed to elevated temperature suggests that malate should also be reduced. Malate deple‐
tion implies that less carbon is redirected to mitochondria and that there is less energy
available for N2 fixation. The observed decrease in enzyme activity could be related to a
reduction of the total soluble protein of the bacteroid. The large (70%) reduction in solu‐
ble protein could affect nitrogenase protein responsible for N2 fixation activity. These re‐
sults show that the nodule bacteroid fraction was more sensitive to drought and elevated
temperature than the nodule plant fraction. Relatively high temperature has also been
shown to influence infection, N2-fixation activity, and legume growth (Mohammadi et al.
2012) and has a strong influence on specific strain and cultivar interactions (Arayankoon
et al. 1990). Reduced nitrogenase activity in such nodules can also be explained on the ba‐
sis of these effects. It has been reported that there is considerable variation in the rhizo‐
bia strains in terms of survival and nodulation ability at high temperature (Michiels et al.
1994). Karanja and Wood (1988) found that a high percentage of the strains that persisted
at 45 °C lost their infectiveness. They attributed these losses to plasmid curing. It appears
that every legume and Rhizobium combination has an optimum temperature relationship,
which is around 30 °C for clover and pea, between 35 to 40 °C for soybean, peanut and
cowpea, and between 25 to 30 °C for common bean (Long 2001).
3.2. Desiccation
3.2.1. Survival of Rhizobia
One of the most severe and widespread problems facing the crops production is the degra‐
dation of soil quality due to desiccation and salinity. In fact, almost 40% of the world’s land
surface is affected by salinity-related problems (Vriezen 2007). These two harsh environmen‐
tal conditions can have a dramatic impact on the endogenous soil bacteria (Fierer et al. 2003;
Han et al. 2005). Water, and its availability, is one of the most vital environmental factors to
affect the growth and survival of micro-organisms (Potts 1994). Desiccation is one of the
most common stresses soil microorganisms have to face so often. The responses of bacterial
cells to desiccation can be: shrinkage of the bacterial cytoplasm and capsular layers, increase
in intracellular salt levels, crowding of macromolecules, damage to external layers (pili,
membranes), changes in ribosome structure, and decrease in growth. Reactive oxygen spe‐
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cies can also damage proteins and DNA, leading to accumulation of mutations (Potts 1994).
Death of rhizobial cells during desiccation was suggested to associate with changes in mem‐
brane integrity (Bushby and Marshall 1977). It has been hypothesised that during dehydra‐
tion, the removal of water hydrogen bond to the phospholipid head groups of the
membrane decreases the spacing between adjacent lipids. Then, the membrane is converted
from the liquid crystalline into the gel phase already at room temperature. Subsequent rehy‐
dration results in a further phase transition of the membrane back to the liquid crystalline
phase. As consequence, the membrane barrier is disrupted, leading to leakage of mem‐
branes (Potts 1994: Welsh 2000). Desiccation tolerance is the ability of cells to undergo nearly
absolute dehydration through air drying, without being killed. This is the most severe water
deficit stress since the removal of the cell-bound water imposes such structural, physiologi‐
cal and biochemical stresses that cells must adapt or die (Billi and potts 2002).
Of particular importance to the crop production is the impact of these harsh environmental
conditions on the persistence and survival of rhizobia. The ability of rhizobia, to survive
desiccation depends on their ability to cope with radiation stresses, reactive oxygen species,
certain salts and solutes, and temperature extremes (Potts 1994; Billi and potts 2002; Ramos
et al. 2001). Desiccation stress can be differentiated into three main stages: drying, storage
and rehydration. These phases can be manipulated in several ways, namely, by the severity
and the speed of drying and rehydration and by the duration of storage. The consequences
of drying are fourfold: (i) the accumulation of salts and solutes, (ii) hyperosmotic stresses,
(iii) the distribuance of metabolism when a certain water activity has been reached, and (iv)
the accumulation of damage when the aqueous monolayer is removed from macromole‐
cules. The accumulation of damage during storage is comparable to that caused by ionizing
and UV radiation and damage by reactive oxygen species (Mattimore and Battista 1996;
Shirkey et al. 2000) when organisms are not metabolically active and thus unable to repair
any damage (Potts 1994). Finally, during rehydration, hypoosmotic stresses and the appear‐
ance of reactive oxygen species affect survival (Stead and Park 2000). A low survival of rhi‐
zobia occurs when drying was rapid (Bushby and Marshall 1976; Antheunissen and
Arkestein-Dijksman 1979). These results were confirmed by Mary et al. (1985, 1986) who
used Sinorhizobium meliloti RCR2011 to test survival after slow and fast drying with and
without the addition of salts. It was recorded that fast drying process resulted in a decrease
in survival of rhizobia in mineral soil (Chao and Alexander 1984). The reduction of water
content causes the accumulation of salts and other compounds that produce osmotic and
salt stress. High concentration of these compounds may be toxic and decrease the viability
of cells (Steinborn and Roughley 1975; Vriezen et al. 2006). Conversely, the accumulation of
certain compounds, including osmoprotectants and compatible solutes, may increase desic‐
cation survival (Fougere and Le Rudulier 1990; Gouffi and Blanco 2000; Madkour et al.
1990). Osmoprotectants are exogenous solutes that stimulate bacterial growth in an environ‐
ment with high osmolality, whilst compatible solutes are specific organic osmolytes that ac‐
cumulate in high amounts within a cell to counter a hyper-osmotic gradient, but do not
conflict with cellular functions (Miller and Wood 1996). Several compatible solutes have also
been shown to stabilise enzyme stability in cells under stressful conditions (Poolman et al.
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2002). Some compounds can function as osmoprotectants and compatible solutes, whilst
some can only function as one of these groups.
Many osmoprotectants are transported into the cytoplasm where they act as, or are convert‐
ed into compatible solutes (Hirsch 2010). Compatible solutes can be collected in high con‐
centrations (Bremer and Krämer 2000). Since only a limited number of compounds meet the
required criteria, the same compatible solutes are employed against hyper-osmosis through‐
out various bacteria (Braun 1997). Different compatible solutes work more effectively than
others within their bacteria; e.g. glycine betaine is more effective in S. meliloti and E. coli than
it is in Bacillus subtilis (Botsford and Lewis 1990); whilst proline is a compatible solute in E.
coli but not in rhizobia (Gloux and LeRudulier 1989). Also, the strength of hyper-osmolarity
can determine how the bacteria respond and what osmoprotectants are used (Breedveld et
al. 1990; Gouffi et al. 2000). In a similar way, the compound used to bring about hyper-os‐
mosis can stimulate a stronger stress response compared to others; e.g. generally sodium
chloride (NaCl) induced hyper-osmosis causes a stronger stress response then sucrose in‐
duced hyper-osmosis, due to the ionic nature of NaCl (Gloux and Le Rudulier 1989). Com‐
patible solutes can either by synthesized de novo, when required by the bacteria, or they are
accumulated from the environment, depending on the situation. Under conditions where os‐
motic upshift is severe and immediate, cells do not have the time required to synthesise
compatible solutes and so must acquire them from their environment. Reduction of water
activity below 0.53 inhibited the function of RNA polymerase (Brown 1990). The storage
phase is characterized by a slow decline in viable counts in rhizobia after slow drying. Mary
et al. (1985, 1986) noted that the better survival of Sinorhizobium during storage under desic‐
cation conditions from 22% to 67% than at 3% and 83.5% relative humidity. Similar trend
have been recorded in Bradyrhizobium (Mary et al. 1994; Boumahdi et al. 1999). Cell death
occurs at 83.5% relative humidity due to failure of intracellular enzymes. Antheunissen et al.
(1981) showed that when dried occur at slow rate, rhizobia can survive desiccation for up to
4 years. These long-term storage studies are rare, but they show that in the family Rhizobia‐
ceae, sinorhizobia can survive desiccation for years. A decline in viable cells during longterm storage under desiccation conditions can be explained by the accumulation of oxygenand radiation-induced damage (Vincent et al. 1961; Mary et al. 1994). The rate of rehydration
has important consequences for repairing of accumulated damage and survival of bacteria.
Combined process of drying and fast rehydration resulted in rupture of the cell envelope.
Cells ruptured on the subpolar region which is also the region from which flagella originate.
A suggestion is made that the point where a flagellum emerges from the cell is a point of
weakness (Bushby and Marshall 1977; Salema et al. 1981). Slow rehydration of S. meliloti, R.
leguminosarum, and Pseudomonas putida from dried inoculant formulations provide higher
viable counts than did rapid rehydration (Kosanke et al. 1991). Chen and Alexander (1973)
reported that a higher percentage of drought-tolerant than drought-sensitive bacteria was
able to grow at low water activities. When these bacteria were grown in media with high
salt concentrations, bacteria generally became more tolerant of prolonged drought and they
persisted longer. In general, rhizobia do not, or cannot, synthesise their own solutes so use
uptake systems to accumulate them (Gloux and Le Rudulier 1989). However, Streeter and
Gonez (2006) reported that many rhizobia accumulate various carbohydrates such as treha‐
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lose, a disaccharide made up of two glucose molecules joined together by an α, α-1,1 link‐
age, is employed by many organisms to protect membranes and proteins from desiccation
stress. Rhizobia also accumulate trehalose, among other carbohydrates, and also betaine and
proline, in response to desiccation. Trehalose and sucrose are the only carbohydrates that
are synthesized de novo in response to stress. Bacteria can produce trehalose from glucose
6-phosphate and UDP-glucose (the OtsA–OtsB pathway), from glycogen-like α(1-4)-linked
glucose polymers (the TreY–TreZ pathway) and from maltose (the TreS pathway). The
TreYZ pathway is common to many rhizobia (Streeter and Bhagwat 1999), whereas the Ot‐
sAB and TreYZ pathways are found in R. leguminosarum bv. trifolii strain NZP561 (Mcintyre
et al. 2007). Cultured Bradyrhizobium japonicum USDA110 and B. elkanii were found to have
three enzymes for trehalose synthesis: trehalose synthase (TS), maltooligosyltrehalose syn‐
thase (MOTS), and trehalose-6-phosphate synthetase (Streeter and Gomez 2006). Trehalose
at relatively high concentrations is also present in B. japonicum bacteroids residing within
nodules, suggesting that these differentiated nitrogen-fixing cells are under stress. Addition
of trehalose to culture medium of B. japonicum at the time of desiccation stress had a signifi‐
cant positive effect on survival (Streeter 2003). Although it may not be practical to use treha‐
lose as a carbon source in inoculant production, it may be possible to engineer greater
trehalose accumulation in rhizobia to enhance their survival in response to dryness. Treha‐
lose may protect desiccated cells by their ability to form glasses under dry conditions, in this
way maintaining the native conformation of proteins and other macromolecules (Ramos et
al. 2001). Trehalose levels also increase in R. leguminosarum bv. trifolii TA1 cells as they en‐
counter osmotic stress (Streeter 1985; Breedveld et al. 1993). R. leguminosarum bv. trifolii
strain NZP561 accumulates trehalose upon entry into stationary phase (Mcintyre et al. 2007),
but in this rhizobial strain, trehalose synthesis is constitutive and modified post transcrip‐
tionally rather than induced as in other rhizobia. Mutations in otsA or treY individually in
strain NZP561 did not dramatically affect trehalose accumulation, but double otsA treY mu‐
tants did not accumulate trehalose and were more sensitive to desiccation. They were also
less competitive with regard to occupying nodules than were wild-typestrains (Mcintyre et
al. 2007). The transcriptional analysis of the genome of B. japonicum subjected to desiccation
stress indicated that genes critical for pilus are upregulated (Cytryn et al. 2007). Pili, espe‐
cially type IV pili, are often important for biofilm formation (Shime-Hattori et al. 2006; Jurci‐
sek and Bakaletz 2007). It is extremely likely that desiccation-stressed B. japonicum cells
show some of the same patterns of gene expression, as do cells in biofilms. Similarly, S. meli‐
loti cells grown under salt and osmotic stress (Dominguez-Fererras et al. 2006) upregulate
some of the same genes uncovered in transcriptome arrays of biofilm cells of other bacteria
(An and Parsek 2007). Biofilms are one of the many ways that bacteria use to protect them‐
selves from desiccation and it is well known that exopolysaccharide, an important compo‐
nent of the biofilm matrix, protects bacteria from drought stress. Loss-of-function EPS
mutants of many bacteria show impaired biofilm formation (Yildiz and Schoolnik 1999; Da‐
nese et al. 2000; Whiteley et al. 2001; Matsukawa and Greenberg 2004), as do S. meliloti exoY
loss-of-function mutant cells (Fujishige et al. 2006) and exopolysaccharide mutants of M.
tianshanense (Wang et al. 2008). Nevertheless, it is not known whether exopolysaccharide-de‐
ficient mutants that are incapable of biofilm formation are less capable of surviving desicca‐
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tion stress under field conditions. Vanderlinde et al. (2010) demonstrated the important role
for exopolysaccharide in desiccation tolerance in Rhizobium leguminosarum bv. viciae 3841
and identify a novel genetic element (ABC transporter) required for biofilm formation. Iden‐
tification of a novel genetic element required for desiccation tolerance, and proper biofilm
formation. An increased tolerance to desiccation can enhance the survival of rhizobacteria
within the soil considerably (Rokitko et al. 2003). This is of particular interest with nitrogenfixing rhizobia because desiccation is a major cause of the poor on-seed survival rates of
commercial inoculants and the subsequent poor performance of rhizobial inoculants in the
field (Deaker et al. 2004). To further our understanding of how bacteria persist and survive
in the soil environment, and improve on-seed survival of rhizobial inoculants, a complete
understanding of the mechanisms used for desiccation tolerance is necessary.
3.2.2. Rhizobium-Legume molecular signalling exchange, noduation and nitrogen fixation
To the best of our knowledge, there is no information available addressing the impact of
drought on molecular signals exchanges between the two partners. Further research is need‐
ed to clarify the impact of drought on Rhizobium-legume molecular signaling exchange.
However, several studies both on herbaceous and woody legumes have shown that drought
cause deleterious effect on nodulation and nitrogen fixation (Singleton and Bohlool 1984;
Zahran and Sprent 1986; Arayangkoon et al. 1990; Marcar et al. 1991; Purwantari et al. 1995;
Hatimi 1999). If the stress conditions are long or/and strong enough, formation of nodules
will cease completely. It is generally believed that the infection process is the most sensitive
phase during nodule development (Singleton and Bohlool 1984). Once nodules have
formed, they are less affected by stress factors than the initial nodule formation process
(Purwantari et al. 1995). Studies with herbaceous legumes having the root hair infection
mode indicated that drought and salinity stresses cause changes in root hair morphology
and decrease the numbers of markedly curling hairs. In mildly water-stressed Vicia faba nod‐
ules, the loss of turgor induced a reduction in the peribacteroid space, the vesicle membrane
became sinous, and the cell cytoplasm appeared granular. Under more severe water stress, a
total disappearance of peribacteroid spaces was associated with the rupture of vesicle mem‐
branes. The bacteroid content appeared to be very heterogeneous (Guerin et al. 1991). A
shortage of water supply can slow the growth of the nodule and accelerate its senescence.
Nitrogenase activity is decreased significantly, accompanied by the decrease in respiratory
activity of the soybean and common bean nodules (Weisz et al. 1985; Gerosa-Ramos et al.
2003). A limitation in metabolic capacity of bacteroids and oxidative damage of cellular
components are contributing factors to the inhibition of nitrogenase activity in alfalfa nod‐
ules (Naya et al. 2007). In addition, the transport of fixed nitrogen out of the nodule is de‐
creased, possibly due to an insufficient supply of photosynthates in stems and leaves under
stress (Huang et al. 1975).
The major pathway of water movement into nodules is through vascular connections with
the root. The surface of the nodule is the main area for gaseous exchange and is therefore
more adapted to water loss than uptake. Nodules always need an efficient water supply to
export the product of fixation. Water-stressed plants transpire at a lower rate than un‐
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stressed plants. A lower rate of water movement out of the nodule during drought stress
may restrict export of N2 fixation products, thus inhibiting nitrogenase activity via a feed‐
back mechanism (Serraj et al. 1999). Water shortage may induce oxidative stress in nodules.
This leads to general decrease of antioxidant activities that are associated with nodule senes‐
cence (Hernandez-Jimenez et al. 2002; Porcel et al. 2003). It was found that water stress im‐
posed during vegetative growth was more detrimental to nodulation and nitrogen fixation
than that imposed during the reproductive stage (Pena-Cabrales and Castellanos 1993). The
mechanisms of desiccation tolerance could be attribitted to the ability to limit to a minimum
the cell metabolism, the increased catalase activity and the presence of specific plasmids for
drought tolerance. Some experiences have shown that working with legumes and rhizobia
strains selected for desiccation tolerance, a symbiotic interaction can take place (Soria et al.
1996). Abd-Alla and Abdel-Wahab (1995b) observed that nodulation and nitrogenase activi‐
ty were significantly decreased by increasing drought stress. Leghaemoglobin and protein
contents of nodule cytosol were also severely inhibited by drought stress. This decline was
attributed to the induction of protease activity. However, carbohydrate contents of the nod‐
ule cytosol increased significantly. This accumulation was attributed to a sharp decline in in‐
vertase activity and low use of sugar by the bacteroids. This study indicated that harmful
effects of water deficits can be alleviated by increasing K+ supplementation (Abd-Alla and
Abdel-Wahab 1995b). Water stress is quickly reflected as changes in hormonal content
(Hsiao 1973). The nodules are an active site of synthesis of auxins and cytokinins. Therefore,
it is likely that nodules, besides the supply of organic N, are a source of cytokinins that
makes the plant more tolerant to water stress (Phillips and Torrey 1973).
Many legumes are very sensitive to excess water. Nodule development and function are
usually more affected than the infection itself, and some effects such as decreased nitroge‐
nase activity may be even more intense than in the case of water deficit. Reduced to zero, the
contribution of O2 to the nodule appears to be the main problem of the effect of waterlog‐
ging. The diffusion of O2 within the nodules is in part regulated by a physical barrier located
in nodular parenchymal cells (Andres et al. 2012). Under stress conditions, the diffusion re‐
sistance increases by identifying a lack of O2 inside the nodule, leading to inhibit its activity
(Day and Copeland 1991). The ability of aerobic bacteria to utilize nitrogenous oxides, as ter‐
minal electron acceptors, enables them to survive and grow during periods of anoxia. This
may be advantageous for the survival of rhizobia in soils (Zablotowicz et al. 1978).
3.3. Salinity stress
3.3.1. Survival of Rhizobia
The world’s demand for food is increasing at such a rate that the ability to meet anticipated
needs in the next several decades is becoming questionable. Irrigated agriculture presently
accounts for about one-third of the world’s production of food and fibre; it is anticipated
that it will need to produce nearly 50 percent by the year 2040 (Rhoades et al. 1999). This
will likely be difficult, because extensive areas of irrigated land have been and are increas‐
ingly becoming degraded by salinization and waterlogging resulting from over-irrigation
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and other forms of poor agricultural management (Ghassemi, et al. 1995). Soil salinity affects
about 800 Mha of arable lands worldwide (Munns and Tester 2008), and this area is expand‐
ing. Salinity affects agricultural production in arid and semiarid regions, where rainfall is
limited and is not sufficient to transport salts from the plant root zone (Tester and Daven‐
port 2003). The damaging effects of salt on organisms are caused not only by osmotic forces,
but also by toxic levels of sodium and chloride. Salinity stress is important factor limiting
the productivity of leguminous crops. Soil salinity reduces survival and growth of rhizobia
in the soil and inhibits rhizobia-legume symbiosis, resulting in lower productivity of le‐
gumes (Abd-Alla 1992; Abd-Alla et al. 1998). Rhizobia are known to be more salt tolerant
than their respective plant partners. Maximal limit of tolerance to salinity is superior in rhi‐
zobia as compared to their host plant which frequently constitute the limiting factor in sal‐
ine soils (Kassem et al. 1985). In some cases bacteria that are highly salt tolerant as freeliving cells produce ineffective nodules, showing reduced symbiotic efficiency and/or low
rates of nitrogen fixation (Chien et al. 1992, Abd-Alla and Abdel-Wahab 1995a). Some bacte‐
rial strains of various species are able to grow within the range of 300–700 mM NaCl (Mpe‐
pereki et al. 1997). Many authors reported that some rhizobial strains can persist and
survive in the saline soils (ElSheikh and Wood 1989, 1990a; Ishaq et al. 1989), although the
majority of rhizobia are not capable of tolerating the harmful effects of high osmolarity
(Soussi et al. 2001). The ability of rhizobia to tolerate salt stress depends also on the species
and even the strain of rhizobia studied (Bernard et al. 1986). Fast growing rhizobia are gen‐
erally considered to be more tolerant to saline stress than bradyrhizobia (ElSheikh and
Wood 1990b) and strains isolated from saline soils are typically more tolerant (Hua et al.
1982). Nevertheless, many fast growing rhizobia are very salt sensitive and some rhizobia
isolated from saline soils are sensitive (Zahran 1999). The ability of rhizobia to adapt to fluc‐
tuations in the osmolarity of their surrounding is of fundamental importance for their sur‐
vival. Osmotolerant rhizobia use a variety of accumulated or non-accumulated
osmoprotectants, including betaines, amino acids and sugars as a strategy to counter the
high oscillations of their environment osmolarity (Bernard et al. 1986; Gouffi et al. 1999). Os‐
moregulation is the main strategy employed by rhizobia to cope with salt stress (Ghittoni
and Bueno 1996), including altered polysaccharide production upon salt treatment (Lloret et
al. 1998). Survival and growth in saline environments are the result of adaptive processes,
such as ion transport and compartmentation, osmotic solute synthesis and accumulation,
which lead to osmotic adjustment and protein turnover for cellular repair (Munns and Ter‐
maat 1986; Paul and Cockburn 1989).
Bacteria grown in saline environments must maintain positive turgor across the membrane
by allowing influx of salt or solutes or by exclusion of salts via the production of compatible
solutes or other organic osmolytes. Bacteria prefer uptake over de novo synthesis of organic
osmolytes when present in surrounding environment. Exogenous osmolytes that improve
cell growth under adverse osmotic conditions are referred to osmoprotectans (Galinski
1995). Under elevated salinity S. meliloti can accumulate K+ ions (Miller and Wood 1996) and
an organic anion, glutamate, and synthesize the following compatible solutes: N-acetylglu‐
taminylglutamine amide (NAGGN), trehalose, and glycine betaine, if the medium contains
its precursor, choline (Hua et al. 1982; Botsford and Lewis 1990; Smith et al. 1994; Talibart et
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al. 1994, 1997). Recent data indicate that the set of endogenous osmolytes produced by rhi‐
zobia can vary at least according to the species level and NAGGN has been found only in S.
meliloti (Smith and Smith 1989; Smith et al. 1994; Talibart et al. 1994, 1997). The types of the
accumulating osmolytes also depend on the stress level and on the growth phase of the cell
culture. In S. meliloti, glutamate accumulated at low salt concentrations, but at higher levels,
glutamate and NAGGN were observed. All three osmolytes, glutamate, NAGGN, and treha‐
lose accumulated only at extremely high NaCl concentrations (Smith et al. 1994). When gly‐
cine betaine was exogenously supplied in the growth medium, it accumulated as the major
osmolyte during the lag and early exponential phases, whereas glutamate and NAGGN pre‐
vailed at the late exponential phase (Talibart et al. 1997). There was no improvement in the
growth of chickpea rhizobia with the addition of glutamate to 340-400 mmol-1 NaCl in the
medium (Botsford 1984; Elsheikh and Wood 1989; Gonzalez-Gonzalez et al. 1990).
• Trehalose was the major osmolyte of the stationary phase (Smith et al. 1994, Talibart et al.
1997). A number of different environmental factors, such as the level of osmotic stress,
growth phase of the culture, carbon source and osmolytes of the growth medium control
the combination of naturally occurring endogenous osmolytes in rhizobial cells (Smith et
al. 1990, 1994). Bacteria prefer uptake of compatible solutes over synthesis de novo. The
transport systems for external osmolytes (osmoprotectants) are relatively unspecific, and
bacteria accept components of plant and animal origin (Galinski 1995). The enhancement
of growth of rhizobia from different species, resulting from added glycine betaine and
other betaines under saline conditions, has been recorded (Sauvage et al. 1983, Bernard et
al. 1986). S. meliloti and many other rhizobial species can use glycine betaine as a C and/or
N source under low osmotic stress but as an osmoprotectant in high osmolarity. Contrary,
Bostford (1984) observed that exogenous application of glycine betaine did not ameliorate
inhibition of the growth of S. meliloti in NaCl. Only the slow-growing B. japonicum, which
is considered as an osmosensitive species, is incapable to transfer glycine betaine and its
precursor choline (Boncompagni et al. 1999). Under low osmotic stress glycine betaine is
degraded via successive demethylation to glycine, while at elevated osmolarity the catab‐
olism is blocked and glycine betaine is accumulated in cells (Sauvage et al. 1983; Bernard
et al. 1986; Smith et al. 1988; Boncompagni et al. 1999). Furthermore, Exogenous applica‐
tion of calcium alleviated the inhibitory effect of NaCl on growth of rhizobia and induced
NaCl tolerance (Chien et al. 1991; Howieson et al. 1992; Reeve et al. 1993). There is an in‐
creasing demand for identifying rhizobial species that could also work under stressed soil
environment so that the productivity of the inoculated legumes does not suffer under der‐
elict soils.
3.3.1.1. Rhizobum-legume signalling exchange
Nodulation (nod) factors are products of nod gene exerssion of rhizobia. Nod factors are sig‐
nals known to be used in the communication with leguminous plants in the process of root
nodule formation. Nod factors are major host-specificity determinants (factors that deter‐
mine which rhizobium nodulates with a specific host plant) and trigger the nodulation pro‐
gram in a compatible host (Mergaert et al. 1997). The influence of salt (NaCl) stress on the
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expression of enzymes involved in Nod factor production, and on the huge variety of Nod
factors produced by growing R. tropici have been investigated (Estevez et al. 2009). In sur‐
prising study by Guasch-Vidal et al. (2013) observed that, in the absence of flavonoid induc‐
ers, high concentrations of NaCl induced nod genes and the production of Nod factors. The
higher transcriptional activity at the nod region in the presence of NaCl, as revealed by the
increased the β-galactosidase activity of a nodP:lacZ fusion, that lead to a higher and detecta‐
ble production of Nod factor. However, a high concentration of salinity had adverse effect
on Nod factor activity and decreased root hair deformation in soybean (Duzan et al. 2005).
Evidence existed that introduction of exogenous nod gene inducers increases nodulation and
nitrogen fixation of some legume species (Abd-Alla et al. 2013a). Pre-treatment of Bradyrhi‐
zobium japonicum with genistein increased nodulation and nitrogen fixation of soybean and
common bean (Zhang and Smith 1996; Abd-Alla 2001, 2011); and pre-induction of Rhizobium
leguminosarum with hesperetin and naringenin was found to stimulate nodulation and plant
dry matter accumulation of pea and lentil plants (Begum et al. 2001). Flavonoid inducers act
in low concentrations and the pre-activation of rhizobia used as inoculants (biofertilizers) in
undoubtedly economically justified. Pre-activation of strains might increase rhizobial com‐
petitiveness in the soil environment (Hungria and Philips 1993). Indeed, it has been shown
that flavonoid pre-activated B. japonicum increased soybean nodule quantity and weight
(about 30%), the seasonal level of N2 fixation (35%) and yields (10-40%) when compared to
conventional inoculants (Zhang and Smith 2002). Likewise, field pea and lentil plants dis‐
played increased nodulation and biomass production when inoculated with Rhizobium legu‐
minosarum preinduced with hesperetin (Begum et al. 2001). In common bean (Phaseolus
vulgaris L.), a negative effect of NaCl on the expression of nod genes by Rhizobium tropici and
Rhizobium etli and on nodulation factors’ pattern was observed (Dardanelli et al. 2008). The
preincubation of B. japonicum with the signal molecule genistein, under saline conditions,
was described as a method to alleviate the stressful effects of salt on soybean–B. japonicam
symbiosis (Miransari and Smith 2009). Efforts to overcome the inhibitory effect of salinity on
nodulation of feungreeck by pre-inocubation of Rhizobium tibeticum with hesperetin and
apaginein were successful (Abd-Alla et al. 2013a). It is supposed that flavonoids have selec‐
tive value in plant-microbe interaction under salinity stress. Improving legume inoculation
efficiency is extremely important to improve legume production under drastic conditions.
3.3.1.2. Rhizobium-legume nodulation and nitrogen fixation
In legumes salinity can result in a root system devoid of root hairs and a mucilaginous layer,
and incapable of forming an infection thread (Singleton and Bohlool 1984; Zahran and
Sprent 1986). Unsuccessful symbiosis may be attributed to failure in the infection process
due to poor establishment of Rhizobium (Rai and Prasad 1983). The reduction of nodulation
in soybean under saline conditions was attributed to shrinkage of the root hairs (Tu 1981).
Yousef and Sprent (1983) showed that NaCl affected nodulation and they concluded that
there may also be effects on infection. In salt-treated faba bean plants, total nodule weight
per plant was reduced with increasing amounts of salt given. A reduction of 16.7, 4l.2, 50.8
and 72.6% in nodule weight was caused by salinity levels of 5.8, 8.8, 1l.6 and 14.6 dSm-l, re‐
spectively. Total nodule weight per plant followed a similar pattern to nodule number per
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plant (Abd-Alla 1992). The detrimental effects of salinity on nodulation parameters were
most pronounced for NOD1-3 and Williams 82, intermediate for DR-1, and less marked for
PI416937. Self-grafted NOD1-3 plants showed 50 to 62% inhibition in nodulation responses
(activity, number, mass) while grafting of PI416937 scions to NOD1-3 roots resulted in less
than 7% inhibition by salt. Nodule number on PI416937 roots was greater when grafted to
NOD1-3 scions (relative to self-grafted PI416937 plants), confirming a shoot role in autore‐
gulation of nodule number (Abd-Alla et al. 1998). These results indicated that shoot factors
are of primary importance in determining salt-tolerance of the PI416937 genotype and that
hypernodulation expression in the mutant is negatively affected by salt treatment.
The salt injury on the symbiotic interaction not only inhibits the formation of the nodules,
but also thereby leads to the inhibition in nitrogenase activity and reduction of the growth
of the host plant. Other effects of salinity on the nodulation, includes formation of non-func‐
tional nodules with abnormal structure, and degradation of peribacteroid membrane (Bola‐
nos et al. 2003). Tu (1981) observed that the interaction of Bradyrhizobium japonicum to the
root hairs under increasing concentrations of NaCl in the range from 0% to 1.8% in the cul‐
ture solution was affected. At a concentration of 1%, inhibition was evident in the bending
of the hairs, a phenomenon markedly accentuated as rising concentrations. At 0.2% NaCl,
growth was slow and at 1.2% nodulation did not occur. Results of some experiments on Vi‐
cia faba and Vigna unguiculata showed that the application of different concentrations of
NaCl at the time of root hair formation significantly reduced root colonization, root hair
curling, shrinking root hairs and hypodermic cells (Zahran and Sprent 1986; Georgiev and
Atkins 1993). Several hypotheses have been suggested to explain the negative effects of salt
on nitrogen fixation in plant legumes. It is likely that the depression in specific nitrogenase
activity was due to salt reducing the protein, leghaemoglobin and carbohydrate contents of
both the cytosol and the bacteroids. Salinity levels at 11.6 and 14.6 dSm-1 induced a 37 and
59% decline in carbohydrate and 41 and 53% in leghaemoglobin and 19 and 53% in protein
content of nodules, while nodule C2H2 reduction (specific nitrogenase activity) showed a 55
and 61% decreases, respectively (Abd-Alla 1992). Abd-Alla and Omar (1997) reported that
0.5% NaCl significantly reduced nodulation of fenugreek and addition of wheat straw and
cellulytic fungi minimize the adverse effect of salinity on nodulation.
Protein synthesis is readily inhibited by water stress because of the decreases in the level of
polyribosomes (Bewley 1981). Thus, the decline of nodule soluble protein may result from a
general reduction of protein synthesis and from an increased protease activity in the cytosol
(Becana et al. 1986). The disappearance of nodule soluble protein induced by plant water
stress can be compared to the situation naturally occurring during nodule senescence (Pla‐
dys and Rigaud 1985) or after feeding the plant with nitrate (Ohyama and Kumazawa 1981,
Nishiwaki et al. 1997; Ohyama et al. 2011; Abdel-Wahab and Abd-Alla 1995a,b, 1996). In
both cases, the presence of active proteases was responsible for the protein digestion, and it
seems likely that these enzymes may also be induced under water restricted conditions
(Guerin et al.1990; 1991). It is clear that most biochemical parameters of the bacterial and
plant components of the nodules (cytosol) were affected by high levels of salinity of 100 and
125 mM NaCl (Abd-Alla 1992). Both the protein and carbohydrate contents of nodule cyto‐
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sol were more sensitive to salinity than the corresponding bacteroids at the same levels of
salinity. The negative effects of salinity on the oxygen diffusion barrier, which normally
would reduce the O2 flux into the nodule was also demonstrated (Soussi et al. 1998; Serraj
and Drevon 1998). The decline in nitrogen fixation can be attributed to a reduced carbon
supply to bacteroids, mainly in the form of malate limitation and likely a result of the salt
induced inhibition of nodule carbon metabolism through the inhibition of sucrose synthase
activity (Ben Salah et al. 2009). The accumulation of solutes such as proline, sucrose, and Dpinitol has been described in nodules of some legumes such as alfalfa and cowpea, and they
exert an osmoregulatory function in situations of salinity (Irigoyen et al. 1992). Miransari
and Smith (2007) indicate that under high levels of stress plant spent most of its energy to
inhibit or alleviate the stress rather than developing a symbiosis with N-fixing bacteria. Mir‐
ansari and Smith (2008) reported that genistein application into rhizobia inoculant improves
plant growth through improved nodulation and nitrogen fixation in both normal and salt
stress conditions. Inhbition of N2-fixation of legumes under salt stress seems to some extent
to be due to nodule senescence. More work is required to elucidate the mechanisms by
which salinity interferes with normal nodule physiology and function to alleviate the inhibi‐
tory effect of salinity in crop legume production.
3.4. Soil pH
3.4.1. Survival of Rhizobia
Agricultural soils are either alkaline or acidic that effect on rhizobial growth, survival and
subsequent formation of nitrogen-fixing symbiosis with a legume host. Global warming and
agricultural practices cause increase in the amount of soil affected by acidity, and thus limit
legume crop productivity. Worldwide, more than 1.5 Giga hectares of acid soils limit agri‐
culture production (Graham and Vance 2000) and as much as 25% of the agriculture land is
impacted by problems associated with soil acidity (Munns 1986). Each Rhizobium has its own
optimum pH, under which it grows at its best. Although neutral conditions are generally
optimum for bacteria, different species of Rhizobium display varying degrees of pH resist‐
ance as measured by their ability to grow (not just survive) (Glenn and Dilworth 1994). Rhi‐
zobia can be more sensitive to acidic condition than their legume host. Indeed, it is in many
cases the inability of the rhizobia to persist and survive under acidic conditions that reduces
the effectiveness of the symbiosis. Therefore, the selection of rhizobial strains tolerant to
acidic conditions may improve the acid tolerance of the legume through an efficient symbi‐
otic nitrogen fixation under acidity conditions. However, the relationship between low pH
of soil and rhizobia competitiveness, and ability to presist under acidic stress is not always
straight forward. Chen et al. (1993) reported that mutants of R. leguminosarum have been
able to grow at a pH as low as 4.5. Moreover, Foster (2000) recorded that S. meliloti was via‐
ble only below to pH 5.5. Some rhizobia have wide range of pH such as S. fredii can grow
well between pH 4 – 9.5 but B. japonicum cannot grow at the extremes of that range (Fujihara
and Yoneyama 1993). These values are the extremes and hindered the growth and survival
of bacteria between 1 and 2 pH units (Richardson and Simpson 1989). Although some acidtolerant rhizobia strains have been selected (Wood et al. 1988; Vinuesa et al. 2005; Laranjo
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and Oliveira 2011), the mechanisms that employ to survive and grow under acidic condi‐
tions have not been fully elucidated and therefore the molecular basis for differences in pH
toleranceamong strains of rhizobia is still not clear. Several genes, such as actA, actP, exoR,
lpiA, actR, actS, and phrR, were shown to be essential for rhizobia growth at low pH (de
Lucena et al. 2010). Kurchak et al. (2001) have been identified 20 genes in R. leguminosarum
that are responsible for acid stress namely as act genes (acid tolerance).
Root nodule bacteria employ mechanisms for maintenance of intracellular pH (pHi) are cru‐
cial. Like other Gram-negative bacteria, root nodule bacteria show an adaptive acid toler‐
ance response, with growth at moderately acidic conditions protecting against an extreme
acid shock. Variations in acid tolerance within species of root nodule bacteria imply a genet‐
ic basis to low pH tolerance and studies of acid-sensitive mutants suggest that as many as 20
genes could be involved (Glenn and Dilworth 1994). Sensing mechanisms are composed of
two components: a sensor and a regulator, and one has been found in S. meliloti; the genes
actR and actS encode for the regulator and sensor respectively (Tiwari et al. 1996b). ActS is
the membrane bound product of actS that, on detection of external acidity, activates ActR
(product of actR) via phosphorylation. ActR then goes on to activate the transcription of oth‐
er acid response genes within the bacterium (Tiwari et al. 1996b). The membrane bound
product of actA is basic and responsible for maintaining internal pH at around 7, when the
external pH drops below 6.5 (Tiwari et al. 1996a). They demonstrated that mutants defective
in this gene are unable to maintain intracellular pH and cannot grow at a pH lower then 6.
Tiwari et al. (1996a) indicated that calcium involves in acid tolerance mechanism S. meliloti.
Riccillo et al. (2000) reported that glutathione play a key role in acid tolerance of Rhizobium
tropici. Glutathione also provides protection against chlorine compounds in E. coli and
against oxidants in E. coli and Rhizobium leguminosarum bv. phaseoli (Crockford et al. 1995).
The activation of glutathione synthesis might be essential for tolerance to acid stress (Muglia
et al. 2007). TypA is act as a regulator by controlling the phosphorylation of proteins and
required for growth at acidic condition (Kiss et al. 2004). Reeve et al. (1998) show that, in
addition to the genes like actA, acts and actR that are absolutely essential for growth of S.
meliloti at low pH, there is phrR gene which, while not essential for growth, appears to be
induced by exposure to low pH. Cunningham and Munns (1984) found that Rhizobium pro‐
duce greater amounts of exopolysaccharides are able to survive in acidic conditions more
successfully than Rhizobium that can only produce smaller amounts.
Many agricultural fields are alkaline with an average pH above 7.0 to 8.5. A major problem
in alkaline soils is reduced nutrient availability. Alkalinity stress can also retard Rhizobium
from growing and subsequent establishment of a viable nitrogen-fixing symbiosis with a le‐
gume host. Therefore, it makes good sense agriculturally to select rhizobial isolates that are
tolerant of alkaline conditions as well as capable of nodulating legumes (Abd-Alla et al.
2013b). Although R. leguminosarum bv. trifolii has been reported to colonize soil at a higher
rate and produce nodulates at a higher frequency in alkaline conditions; it is also known to
grow unaffected at pH 11.5 (Zahran 1999). Homospermidine, a polyamine present in high
concentrations in root nodule bacteria, is also known to accumulate in B. japonicum in alka‐
line conditions, although its function is unknown (Fujihara and Yoneyama 1993). Rhizobium
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etli strains EBRI 2 and EBRI 26 are more competitive than strain CIAT 899G in soils with
high salt or alkaline conditions (Shamseldin and Werner 2004). These isolates can be use as
inoculants for alkaline agriculture fields.
3.4.1.1. Rhizobium-legume molecular signalling exchange
Soil pH adversely affects several stages during the development of symbiosis, including the
exchange of molecular signals between the legume and the microsymbiont (Hungria and
Vargas 2000). Hungria and Stacey (1997) observed that release of isoflavonoids nod-gene in‐
ducers by soybean and common bean roots was less at pH 4.5 than at pH 5.8 with some
nodulation genes, including nodA, switched off as the pH decrease (Richardson et al. 1988 a,
b). At low pH, induction of nod gene expression in R. leguminosarum biovar trifolii is mark‐
edly reduced in the presence of flavone-inducer. Furthermore, inducibility of nod gene ex‐
pression in R. leguminosarum bv. trifolii is also affected by a net reduction in the
concentration of nod gene-inducing factors present in the root exudates of clover seedlings
grown in acidic conditions (Richardson et al. 1989). Depression in soybean and common
bean nodulation under acidic conditions may be partially alleviated by supplementation
with flavonoid nod-gene inducers (Hungria and Stacey 1997), with studies to examine varie‐
tal differences in response or to maximize the expression of rhizobial nod-genes under acid
conditions still needed. Also, acidic soil can affect the production and excretion of nodula‐
tion factors of R. leguminosarum bv. trifolii (McKay and Djordjevic 1993). Soil pH has been
shown to effect on the profile of Nod factors secreted by R. tropici CIAT899, which is tolerant
of acidic conditions (Morón et al. 2005). At least seven different classes of Nod factor struc‐
tures were identified. More than 50 different Nod factors were detected at pH 4.5, and great‐
er induction of the nod genes also occurred at this pH. This diversity of Nod factor structure
may facilitate nodulation of bean at an acidic pH. Angelini et al. (2003) observed that Initial
root colonization by rhizobia was advesly affected when both acid-tolerant and acid sensi‐
tive (pH 5.0) peanut rhizobia were grown at low pH. This effect could be attributed to defor‐
mation produced by acidic pH on the microsymbiont rather than on host legume. At acid
pH, very low nodC gene expression was observed in acid-sensitive isolates, while a change
in the flavonoids inducer effectiveness was determined in acid-tolerant isolates. There is no
information available on the of effect alkalinity on the exchange of molecular signals be‐
tween the legume and the microsymbiont and additional studies are needed.
3.4.2. Rhizobium-legume nodulation and nitrogen fixation
The availability of some essential nutrients such as calcium, magnesium, phosphorus, and
molybdenum is low in acid soils, whereas high levels of aluminum and manganese can be‐
come toxic to plants and rhizobia (Coventry and Evans 1989). Soil pH can be distorted and
this depending on the type of plant nitrogen nutrition and host legume. Thus, the N2-fixing
plants released protons, which lead to lower the soil pH. The buffering capacity of certain
agriculture field can avoid major changes in pH, but in soils with low cation exchange ca‐
pacity the problem may be significant. It is clear that N2-fixing plants are more sensitive to
acidity than plants of the same species that feed on mineral nitrogen (Andrews 1976).
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Growth of rhizobia in acidified culture media has proved useful for selecting strains with an
ability to colonize the rhizosphere and to nodulate their host plant in acid soils (Cooper
1988). A major problem is to distinguish between the effects of low pH and toxicity of some
minerals, especially aluminum. In acidic soils with pH of >5.0, where heavy metal activity is
relevant, the presence of available aluminum inhibits nodulation.
The information available on alkalinity is scarce compared to that on acidity. Singh et al.
(1973) observed that the number of nodules on alfalfa root can be significantly decreased in
culture solutions containing 0.1% Na2CO3 and NaHCO3. Apparently, soil alkalinity signifi‐
cantly inhibited nodulation and nitrogen fixation of faba bean inoculated with R. leguminosa‐
rum bv. viciae STDF-Egypt 19 (HM587713). However, dual inoculation of R. leguminosarum
bv. viciae STDF-Egypt 19 (HM587713) and AMF improve the nodulation status and nitrogen
fixation of faba bean grown under alkalinity stress (Abd-Alla et al. 2013b). AMF colonization
generally enhances rhizobial nodulation and N2 fixation (Barea and Azcon-Aguilar 1983;
Hayman 1986; Fitter and Garbaye 1994; Chalk et al. 2006). Many authors have now reported
the enhancement of rhizobial nodulation in roots colonized with AM fungi (Meghvansi and
Mahna 2009; Badri et al. 2010; Wang et al. 2011; Sakamoto et al. 2013).
3.5. Pesticides
3.5.1. Suvrival of Rhizobia
Over the past 60 years pesticides have been used increasingly in the environment. Pesticides
are massive applied to agriculture land to produce a larger yield and high quality of crop as
well as reduce the input of labor and energy into crop production (Ayansina 2009). World‐
wide, about 3 billion kg of pesticides are applied each year with a purchase price of nearly
$40 billion per year (Pan-UK 2003). These compounds pose great threats to the varied agroe‐
cosystems. Although the application of these chemicals has been banned or restricted in
many countries especially the developed ones, some developing countries are still using
these compounds because of their low cost and versatility in industry, agriculture and pub‐
lic health (Tanabe et al. 1994, Sarkar et al. 1997). These pesticides can enter the soil environ‐
ment either by direct application or via plant root exudates. This input of pesticides can
affect many soil organisms in different manners. Information from previous studies indicat‐
ed that some soil bacteria are able to tolerate or degrade some pesticides as sole carbon or
nitrogen source (Dick and Quinn 1995; Liu et al. 1991; Sarnaik et al. 2006) bacteriostatic and
lethal effects can also occur (Fox et al. 2007). Some pesticides were toxic and inhibited rhizo‐
bial growth and survival (Graham et al. 1980; Mallik and Tesfai 1983; Singh and Wright
2002; dos Santos et al. 2005).
The effect of pesticides on rhizobia will depend upon the species and strain (Sawicka and
Selwet 1998; Singh and Wright 2002; Zablotowicz and Reddy 2004), type of pesticide ap‐
plied, and the dose applied (Kaur et al. 2007; Zawoznik and Tomaro 2005).
A Rhizobium sp. strain, named PATR, was isolated from an agricultural soil and found to ac‐
tively degrade the herbicide atrazine (Bouquard et al. 1997). They showed that the hydroxy‐
lation of atrazine due to presence of constitutive enzyme consist of four 50-kDa subunits.
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Drouin et al. (2010) showed that fungicides had the highest deleterious effects on the rhizo‐
bia, followed by herbicides and then insecticides. Tolerance or resistance of rhizobia against
pesticides is a complex process and is controlled by physiological/genetic level of microor‐
ganism. These microorganisms developed such tolerance or resistance have the ability to de‐
graded or change the configuration of pesticide (Kumar et al. 1996; Andres et al. 1998a,
Ortiz-Hernández and ánchez-Salinas 2010). The resistance or tolerance against pesticides
might be attributes to physiological activities that induce the microbial metabolism for the
formation of a new metabolic pathway to bypass a biochemical reaction inhibited by a spe‐
cific pesticide (Bellinaso et al. 2003). Dominant resistance of rhizobia to specific pesticide de‐
pends upon genetic modifications, inherited by the subsequent generation of microbes
(Johnsen et al. 2001; Herman et al. 2005). It well established that pesticides not only have
toxic impact on rhizobial growth and survival but also have deleterious effect on their abili‐
ty to produce plant growth promoting substances. Further studies are required to address‐
ing the toxicological effects of pesticides on survival of rhizobial and their plant growth
promoting activates at molecular level to fortify the effective implementation of this ap‐
proach to protect the soil ecosystem from pesticide hazard.
3.5.2. Rhizobium- Legume molecular signaling exchange
Published studies regarding pesticides’ effects on Rhizobium-legume molecular signaling ex‐
change are usually very scattered and done with few pesticides on a limited number of same
genus (Fox et al. 2001, 2004). They reported using in vitro assays that 30 different pesticides
and environmental contaminants specifically disrupted crucial symbiotic signaling between
flavonoid phytochemicals and S. meliloti NodD receptors. Fox et al. (2007) reported in vivo
evidence that a subset of organochlorine pesticides and pollutants inhibit symbiotic signal‐
ing between alfalfa and S. meliloti, resulting in delayed symbiotic recruitment, reduced sym‐
biotic nitrogen fixation, and a decline in alfalfa plant yield. Pesticides may also disrupt
symbiosis by altering the array of flavonoid phytochemicals a plant produces or by reducing
the overall flavonoid secretion pattern, thereby disrupting plant–rhizobial signaling. Pesti‐
cides may affect the plants in a manner that the bacteria-induced root hair deforming fac‐
tors, similar to Nod Rm-1 and related compounds (Truchet et al. 1991), are incapable to
influence morphogenic activity of the plants. Andres et al. (1998b) reported that alfalfa and
soybean seed and root exudates treated with fungicide (thiram) inhibit the expression of rhi‐
zobia nodulation genes. Temporal and chemical specificity of symbiotic signaling is crucial
for coordinating the actions of host legume and microsymbiont necessary for symbiotic ni‐
trogen fixation. In the dynamic soil environment, rhizobia are exposed to a mixture of ago‐
nistic and antagonistic phytochemicals, and the degree to which its NodD receptors are able
to interpret these signals to locate its symbiotic partner. This will most likely determine the
efficiency of symbiotic nitrogen fixation in that particular soil environment.
3.5.3. Rhizobium-Legume and nodulation and nitrogen fixation
Pesticides are essential for controlling plant pests, and accordingly, improve the productivi‐
ty of major crops including legumes. The presence of residual pesticides in the rhizosphere
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may interfere with the infection process by inhibiting bacteria-induced root hair deforma‐
tion and nodule formation. The infection process may be changed either by pesticide effects
on the virulence of the attacking bacteria or by effect on root fibers of plant in which the in‐
fection occurs (Kumar 1981). Nodulation inhibition with pesticides may also be due to alter‐
ation of root hair morphology or to alteration in the quality and quantity of root exudation
(Ratnayake et al. 1978) including isoflavonoid compounds and lectin that play an important
role in the attraction and attachment of rhizobia to root hairs (Hansen 1994). Pesticides ap‐
plied to leguminous crops constitute a potential hazard to growth, nodulation and nitrogen
accumulation (Abd-Alla and Omar 1993). Abd-Alla et al. (2000 b) indicated that root nodule
formation was inhibited in cowpea, common bean and lupin, the effect being most evident
in the case of cowpea. Reduction in the number of nodules on soybean was recorded follow‐
ing trifluralin application at rates of 0.74 and 1.1 kg−1 (Kust and Struckmeyer 1971). Nodule
dry mass of soybean was also decreased in five soil types treated with pesticides (Dunigan
et al. 1972). Nodulation of broad bean was inhibited by the herbicides trifluralin and metri‐
buzin (Bertholet and Clark 1985). Similar results were also obtained by Eberbach and Doug‐
las (1991) using the herbicides paraquat and glyphosate. Abd-Alla and Omar (1993)
recorded a significant reduction in nodule formation by faba bean plants grown in herbi‐
cide-treated pot soil. Curley and Burton (1975) attributed the inhibition in nodulation, con‐
comitant with pesticide application, to the inability of rhizobia/bradyrhizobia to multiply in
the presence of pesticides. Pesticides may inhibit nodulation through their effect on cellulo‐
lytic and pectolytic enzyme production by rhizobia, as occurs in other micro-organisms
(Mahmoud and Omar 1995). Production of these enzymes by rhizobia is essential for root
hair penetration (Hansen 1994). Reports on the effect of agrochemicals on symbiotic attrib‐
utes of legumes are, however, contradictory. For example, Miettinen and Echegoyen (1996)
observed a substantial decline in nodulation in legume crops grown in soil amended with
imidacloprid. Pesticides may influence on the nodule development and effectivenss of nitro‐
gen fixation through the effect inside the host plant or nodule structure (Rup 1988). Aggar‐
wal et al. (1986) evaluated the effect of carbamate on nodulation in Pisum sativum and Vigna
sinensis. They observed that the low doses of the insecticides had little effect on nodulation
whereas the higher concentrations adversely affected it. In a similar study, Alonge (2000)
evaluated the phytotoxicity of imazaquin on the growth of soybean plants and found that
chlorophyll content in the leaves, root nodules, shoot growth, whole plant dry weight, and
grain yield were reduced. Pesticides can reduce the symbiotic efficiency of nitrogen fixing
bacteria and the host leguminous plants. Pesticides, which are either applied to crops or
found as contaminants in the soil, significantly disrupt symbiotic nitrogen fixation and sub‐
sequently lower plant yields. The decrease in the nitrogen content of plant tissue is mainly
due to the side effects of pesticides on rhizobia/bradyrhizobia.The decrease in nodule num‐
ber on pesticide-treated plants may explain the decrease in tissue N (Abd-Alla et al. 2000 b).
Similarly, Anderson et al. (2004) claimed that insecticides (chlorsulfuron) negatively affect
on nodule formation and nitrogen fixation of chickpea. A comparable observation on the ef‐
fect of insecticides (fipronil) on legumes has been reported. For example, the effect of insecti‐
cide fipronil on growth and nodulation of chickpea, pea, lentil and green gram were
determined by Ahemad and Khan (2011a, b).They showed that Fipronil displayed varying
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degrees of toxicity to the tested legumes. The highest toxicity of fipronil was observed on
shoot dry biomass, leghaemoglobin, and chlorophyll content and the seed protein in chick‐
pea, nodule numbers and nodule biomass in pea.
3.6. Heavy metals
3.6.1. Survival of rhizobia
Heavy metals are most important inorganic pollutants such as Cu, Ni, Cd, Zn, Cr, Pb. Heavy
metals enter soil from industrial operation; animal manures and sewage sludge application
after these elements enter to the soil they remain for several thousands of years. Accumula‐
tions of these metals in various ecological systems cause a massive threat to the varied agro‐
ecosystems (Abd-Alla et al. 1999; Ceribasi and Yetis 2001; Cheung and Gu 2007). Recently
there has been increasing concern with heavy metal contamination, not only due to their
toxic impact on living organisms, but also due to their irreversibly immobilized in soil com‐
ponents (McGrath and Lane 1989). Some metals such as Zn, Cu, Ni and Cr are essential mi‐
cronutrients for living organisms at very low level. Optimum level of physiological function
can be achieved in suitable environment and adequate nutrient supplies. Some elements,
such as heavy metals, though essential for organisms, are harmful if present in excess. The
impact of certain metals such as Cd, Hg and Pb on biological and physiological functions of
living organisms is still obscure. However, all these metals could be toxic at relative low
concentrations (Gadd 1992). Soil microorganisms are very sensitive when subjected to mod‐
erate heavy metal concentrations (Giller et al. 1998). High levels of heavy metals in soil
cause dramatic changes in microbial composition and their activities (Baath et al. 1998; Lak‐
zian et al. 2002; Khan and Scullion 2002, Paudyal et al. 2007; Wani et al. 2008a; Khan et al.
2009; Krujatz et al. 2012) resulting in microbial populations with higher tolerance to metals,
but with lower diversity, when compared to unpolluted soils (Baath 1992; Baath et al. 1998).
These changes in ecosystem led to losses in soil fertility. Wood and Cooper (1988) reported
inhibition of multiplication of rhizobial strain caused at 50 µM Al concentration. Al toxicity
is great problem under acidic medium as solubility of free Al ion (Al3+) increases rapidly un‐
der acidic condition (McLean 1976). Fast growing acid producing bacteria, it promotes Al to
show its full toxicity. Since the distribution of various ionic species of Al is pH dependent
(Martin 1991), slight change in pH may significantly affect the relative concentration of the
various charged Al species and their ligands and hence the toxicity of aluminium. Further,
slight change in pH alone can significantly affect the growth of root nodule bacteria (Thorn‐
ton and Davey 1983; Richardson and Simpson 1989). In combination with acidity, Al is detri‐
mental to root nodule bacteria in those cases where growth occurs to longer log periods,
decreases growth rates and lowers final cell densities (Whelan and Alexander 1986). The
toxic effect of aluminium is due to its effect on bacterial DNA (Johnson and Wood 1990;
Martin 1988). The deleterious effects of metals on rhizobial composition within agroecosys‐
tem and different legume genotypes, however, have been contradictory (Wani et al. 2007a,
b, 2008a, b). To validate this concept of conflicting effects of metals on rhizobia, Paudyal et
al. (2007) conducted an experiment which revealed that rhizobia grew poorly in culture me‐
dium supplemented with even lower concentration of aluminium, while rhizobial growth
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was completely inhibited at 50 mM Al concentration (Wood and Cooper 1988; Chaudri et al.
1993; Broos et al. 2004). Hirsch et al. (1993), in a study, demonstrated that the persistence of
R. leguminosarum bv. trifolii was drastically altered by long-term exposure to heavy metals,
and this Rhizobium lost their infectability to form functional nodules. In a similar study,
Chaudri et al. (2000) reported that the irrigation for long term with sewage sludge contain‐
ing Zn or Cu or mixture of Zn and Cu significantly decreased growth and survival of R. le‐
guminosarum bv. viciae and R. leguminosarum bv. trifolii, in soils. On the contrary, Angle and
Chaney (1991) indicated that even in highly contaminated soils, metal activity was not high
enough to exert an antagonistic influence on the soil rhizobial population or the symbiotic
association between alfalfa and R. meliloti. Arora et al. (2010) investigated the impact of alu‐
minium and copper, iron and molybdenum on growth and enzyme activity of fast and
slow-growing rhizobial species. They indicated that Sinorhizobium meliloti RMP5 had a great‐
est tolerance to copper, iron and molybdenum compared to Bradyrhizobium BMP1. However,
these strains were very sensitive to Al than other metals. In addition, Al was significantly
depressed the various enzymatic activities of Sinorhizobium meliloti strains RMP5 and BMP1.
The lack of adverse effects of metals on soil populations of R. meliloti is contrary to several
previous reports with other species of Rhizobium. Many studies indicated that R. leguminosa‐
rum biovar trifoli, or some portion of the symbiotic process, was adversely affected by metal
contamination of soil (McGrath et al. 1988; Giller et al. 1989; Mcilveen and Cole 1974; Rother
et al. 1983). Tong and Sadowsky (1994) found that fast-growing rhizobia were more suscep‐
tible to Zn and Co (20-40 µg ml-1) than slow-growing (80-480 µg ml-1), and less susceptible to
Mo. The discrepancy is related to the differential metal tolerance of the rhizobial species
(Abd-Alla et al. 2012). A higher protein expression was usually related to tolerance mecha‐
nisms in Rhizobium (Saxena et al. 1996, Pereira et al. 2006).
3.6.2. Rhizobium-Legume molecular signalling exchange, noduation and nitrogen fixation
During the last decades, a plethora of reports have been published concerning the impact of
heavy metals on survival of rhizobium and nitrogen fixation. However, there is minor infor‐
mation about the effect of heavy metals on molecular signals between rhizobia and their
host legumes. Recently, Pawlak-Sprada et al. (2011a, b) have found that treatment of soy‐
bean (Glycine max) and yellow lupine (Lupinus luteus) with cadmium (Cd2+) or lead (Pb2+)
trigged an increase of phenylalanine ammonia-lyase mRNA level and phenylalanine ammo‐
nia-lyase activity. Phenylalanine ammonia-lyase is a key enzyme of the phenylpropanoid
pathway. Several products of this pathway such as flavones and isoflavones are strongest
inducer of rhizobial nodulation genes (Firmin et al. 1986; Redmond et al. 1986). The total
amount of isoflavonoids in soybean and yellow lupine were significantly increased by about
15 % in cadmium- and 46 % in lead-treated plants. High concentration of heavy metals may
reduce the isoflavonoid exudate and therefore inhibits the induction of nod genes. Some re‐
ports are available dealing with the toxicity of different heavy metals on nodulation and ni‐
trogen fixation (Bhandal et al. 1990; Hung et al. 1974; Paivoke 1993a, b; Yakolevya 1984).
Conversely, field studies by Heckman et al. (1984) failed to detect adverse changes in either
plant growth or N2-fixation in the sludge-amended soils. Borges and Wollum (1981) added
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
Cd salts to the soil and reported that N2-fixation of soybean was not affected. The deleteri‐
ous effects of high levels heavy metals on nodulation and N2 fixation of Rhizobium–fab bean
symbiosis are probably due to their inhibitory effects on the growth and activity of both
symbionts (El-enany and Abd-Alla 1995). Wetzel and Werner (1995) reported that 75 mg kgsoil as CdCl2 significantly decreased nodulation of alfalfa roots and shoots dramatically in‐
creased. Younis (2007) reported that when 50–200 mg kg-1 soil of Co, Cu, Cd and Zn was
added deliberately to soils used for Lablab purpureus cultivation, these metals significantly
decreased growth, nodulation and nitrogenase activity of plants in both pot and field experi‐
ments. Sepehri et al. (2006) observed that 2 mg Cd/kg soil had a variable effect on symbiotic
properties of S. meliloti strains and consequently on S. meliloti–alfalfa symbiosis. A decreas‐
ing effect of cadmium concentration on root nodules and N concentration in plants inoculat‐
ed with sensitive rhizobial strains in comparison with plants bacterized with tolerant strains
was 68% and 41%, respectively. The effects of metals on rhizobial composition within soil or
nodule environment and different legume genotypes, however, have been contradictory
(Wani et al. 2007a, b, 2008a, b). Mandal et al. (2011) reported that nitrogenase activity was
reduced to almost 2 fold in plants with Arsenate-treated soil but not abolished in root nod‐
ule of blackgram. Their microscopic observations of the cross section of root nodules re‐
vealed that the effective areas of N2-fixing zone were reduced in plants grown in arsenic
contaminated field. Studies examining the effect of Mesorhizobium metallidurans and legumi‐
nous Anthyllis vulneraria symbiosis in soil that was contaminated with Zn, Cd and Pb
showed a significant increase in the total N levels in both plants and soil (Frerot et al. 2006;
Mahieu et al. 2011). Cupriavidus metallidurans is another bacterial species isolated from min‐
ing areas that tolerates high concentrations of heavy metals, and this organism has been
deemed a model of tolerance to metals (Mergeay et al. 2003; Vaneechoutte et al. 2004) and
efficiently nodulates Mimosa pudica (Chen et al. 2008). Recently, a symbiotic capacity for N2
fixation and a high tolerance to heavy metals was demonstrated for some Cupriavidus necator
strains (Ferreira et al. 2012; Silva et al. 2012; Avelar Ferreira et al. 2013).
Metalloprotein (Cd-binding protein) was isolated from nodules of faba bean, grown for 58
days at 100 ppm Cd and 0 Cd by fractionation on a sephadex G-100. The major cadmium
containing protein eluted with a molecular weight higher than 67 kD, close to the void vol‐
ume of the column (200 kD). This protein constituted 38% of the total Cd-binding protein
(metallothioneins). A second peak was noticed in protein fractions of a molecular weight of
about 67 KD. This fraction contained about 53% of Cd-binding protein. A very small amount
of cadmium (ca. 3.4%) was eluted with proteins of 8-17 kD. Apparantly, the cadmium-bind‐
ing protein of 67 kD it appears that Cd-binding proteins play an important role in the detox‐
ification of excess Cd and determining resistance of faba bean-Rhizobium symbiosis to
cadmium toxicity was synthesised only in the presence of appreciable cadmium concentra‐
tions (El-enany and Abd-Alla 1995). The assessment of variation in protein profile is consid‐
ered a good indicator to estimate the level of stress imposed on Rhizobium-legume symbiosis
exposed to heavy metal contamination.
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4. Conclusion
Impact of harsh environmental conditions play an essential role in the control of legume-rhizo‐
bia interactions. They can arrest the growth, multiplication and survival of rhizobia in soil
rhizosphere. The harsh environmental conditions may also have depressive effect on the
steps involved in legume-Rhizobium symbiosis such as molecular signaling, infection proc‐
ess, nodule development and function, resulting in low nitrogen fixation and crop yield. Se‐
lection of hosts and their nitrogen-fixing endosymbionts that are tolerant to a broad range of
environmental stresses is important for agriculture system. Understanding the key molecu‐
lar factors and steps in rhizobia-legume interaction is of crucial importance for the develop‐
ment of Rhizobium strains and legume cultivars with high N2-fixation potential. Prevelence
and aboundance of rhizobia species vary in their tolerance to major environment factors;
consequently, the selection of resistant strains is an important option. Better N2 fixation can
be achieved by selecting tolerance or resistance rhizobia from soil subjected to environmen‐
tal stress. The selection and characterization of harsh conditions-tolerant strains with effi‐
cient symbiotic performance may be a strategy to improve Rhizobium-legume symbiosis and
crop yield in adverse environments. Environmental stress severely affected on various meta‐
bolic activities of legumes including, nod gene expression, photosynthesis, synthesis of pro‐
teins, enzymes and carbohydrates. Therefore, understanding the environmental stress–
rhizobia–legume interactions is urgently required for growing legumes under harsh envi‐
ronmental stress. Research into these areas is currently underway in several research groups
throughout the world and it is anticipated that this research will provide beneficial out‐
comes resulting in improved sustainability and productivity in agricultural systems.
Author details
Mohamed Hemida Abd-Alla1*, Ahmed A. Issa1 and Takuji Ohyama2
*Address all correspondence to: mhabdalla2002@yahoo.com
1 Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut, Egypt
2 Biological chemistry Department, Faculty of Agriculture, Niigata University, Niigata,
Japan
References
[1] Abd-Alla, M. H. 1992. Nodulation and nitrogen fixation in faba bean (Vicia faba L.)
plants under salt stress. Symbiosis 12: 311-319.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[2] Abd-Alla, M. H. 2001. Regulation of nodule formation in soybean-Bradyrhizobium
symbiosis is controlled by shoot or/and root signals. Plant Growth Regulation 34 (2):
241-250.
[3] Abd-Alla, M. H. 2011. Nodulation and nitrogen fixation in interspecies grafts of soy‐
bean and common bean is controlled by isoflavonoid signal molecules translocated
from shoot. Plant, Soil and Environment 57 (10): 453-458.
[4] Abd-Alla, M. H. & Abdel Wahab, A. M. 1995 a. Survival of Rhizobium leguminosarum
biovar viceae subjected to heat, drought and salinity in soil. Biologia Plantarum 37 (1):
131-137.
[5] Abd-Alla M.H. & Abdel Wahab A.M. 1995 b. Response of nitrogen fixation, nodule
activities, and growth to potassium supply in water-stressed broad bean. Journal of
Plant Nutrition 18: 1391-1402.
[6] Abd-Alla, M. H. & Omar S. A. 1993. Herbicides effects on nodulation, growth and ni‐
trogen yield of faba bean induced by indigenous Rhizobium Leguminosarum. Zentralb‐
let fur Mikrobiologie 148: 593-597.
[7] Abd-Alla M.H. and Omar S.A.1997. Wheat straw and cellulolytic fungi application
increases nodulation, nodule efficiency and growth of fenugreek (Trigonella foenumgraceum L.) grown in saline soil. Biology and fertility of Soils 26: 58-65.
[8] Abd-Alla, M. H., Voung, T. D. & Harper J. E. 1998. Genotypic differences in dinitro‐
gen fixation response to NaCl stress in intact and grafted soybean. Crop Science 38:
72-77.
[9] Abd-Alla, M. H., Yan, F. & Schubert, S. 1999. Effects of sewage sludge application on
nodulations, nitrogen fixation and plant growth of faba bean, soybean and lupin.
Journal of Applied Botany 73: 69-75.
[10] Abd-Alla, M. H., Omar, S. A. & Karanxha, S. 2000 b . The impact of pesticide on ar‐
buscular mycorrhizal and nitrogen fixing symbioses in legumes. Applied Soil Ecolo‐
gy 14: 191-200.
[11] Abd-Alla, M. H., Kyro, H. V., Yan, F., Shubert, S. & Peiter, E. 2000 a. Functional struc‐
ture of faba bean nodules: Implications for metabolite transport. Journal of Plant
Physiology 157: 335-343.
[12] Abd-Alla, M. H., Morsy, F. M., El-Enany, A. E. & Ohyama, T. 2012. Isolation and
characterization of a heavy metal resistant isolate of Rhizobium leguminosarum bv. vi‐
ciae potentially applicable for biosorption of Cd (II) and Co. International biodegra‐
dation and Biodeterioration 67: 48-55.
[13] Abd-Alla, M. H., El-enany, A. E., Bagy, M. K. & Bashandy, S. R. 2013a. Alleviating
the inhibitory effect of salinity stress on nod gene expression in Rhizobium tibeticumfenugreek (Trigonella foenum graecum) symbiosis by isoflavonoids treatment. Journal
of Plant Interaction (http://dx.doi.org/10.1080/17429145.2013.824622).
161
162
Advances in Biology and Ecology of Nitrogen Fixation
[14] Abd-Alla, M. H., El-Enany, A. E., Nafady, N. A., Khalaf, D. M. & Morsy F. M., 2013b.
Synergistic interaction of Rhizobium leguminosarum bv. viceae and arbuscular mycor‐
rhizal fungi as a plant growth promoting biofertilizers for faba bean (Vicia faba L.) in
alkaline soil. Microbiological Research (http://www.sciencedirect.com/science/
article/pii/S0944501313001171#).
[15] Abdel Wahab, A. M. & Abd-Alla, M. H. 1995a. Effect of form and level of combined
nitrogen on nitrogenase and nitrate reductase activities in Vicia faba. Biologia Planta‐
rum 37: 57-64.
[16] Abdel Wahab, A. M. &. Abd-Alla, M. H. 1995b. Nodulation and nitrogenase activity
of Vicia faba and Glycine max in relation to rhizobia strain, form and level of combined
nitrogen. Phyton 35: 177-187.
[17] Abdel Wahab, A. M. & Abd-Alla, M. H. 1996. Effect of different rates of N-fertilizers
on nodulation, nodule activities and growth of two field grown cvs. of soybean. Fer‐
tilizer Research 34: 37-41.
[18] AbdelGadir, A. H. & Alexander, M. 1997. Procedures to enhance heat resistance of
Rhizobium. Plant and Soil 188: 93-100.
[19] Aggarwal, T. C., Narula, N. & Gupta, K. G. 1986. Effect of some carbamate pesticides
on nodulation, plant yield and nitrogen fixation by Pisum sativum and Vigna sinensis
in the presence of their respective rhizobia. Plant and Soil 94: 125-132.
[20] Ahemad, M. & Khan, M. S. 2011a. Pesticide interactions with soil microflora: Impor‐
tance in bioremediation. In: Ahmad, I., Ahmad, F. and Pichtel, J. (ed.), Microbes and
Microbial Technology: Agricultural and Environmental Applications. Springer, New
York, p. 393-413.
[21] Ahemad, M. & Khan, M. S. 2011b. Comparative study of the growth parameters of
legumes grown in fipronil-stressed soils. EurAsian Journal of BioSciences 5: 29-36.
[22] Alexandre, A. & Oliveira, S. 2011. Most heat-tolerant rhizobia show high induction of
major chaperone genes upon stress. FEMS Microbiology Ecology 75: 28-36.
[23] Alexandre, A. & Oliveira, S. 2012. Response to temperature stress in rhizobia. Critical
Reviews in Microbiology DOI: 10.3109/1040841X.2012.702097.
[24] Alonge, S. O. 2000. Effect of imazaquin applications on the growth, leaf chlorophyll
and yield of soybean in the Guinea Savanna of Nigeria. Journal of Environmental Sci‐
ence and Health Part B 35: 321-336.
[25] An, D. & Parsek, M. R. 2007. The promise and peril of transcriptional profiling in bio‐
film communities. Current Opinions in Microbiology 10: 292-296.
[26] Anderson, A., Baldock, J. A., Rogers, S. L., Bellotti, W. & Gill, G. 2004. Influence of
chlorsulfuron on rhizobial growth, nodule formation, and nitrogen fixation with
chickpea. Australian Journal of Agricultural Research 55: 1059-1070.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[27] Andres, J. A., Correa, N. S. & Rosas, S. B. 1998a. Survival and symbiotic properties of
Bradyrhizobium japonicum in the presence of thiram: Isolation of fungicide resistant
strains. Biology and Fertility of Soils 26: 141-145.
[28] Andres, J. A., Correa, N. S. & Rosas, S. B. 1998b. Alfalfa and soybean seed and root
exudates treated with thiram inhibit the expression of rhizobia nodulation genes.
Phyton (Vincente Lopez) 62: 47-53.
[29] Andres, J. A., Rovera, M., Guiñazú, L. B., Pastor, N. A. & Rosas, S. B. 2012. Interac‐
tions between legumes and rhizobia under stress conditions. In: Dinesh K. M. (ed.),
Bacteria in Agrobiology: Stress Management. Springer-Verlag Berlin Heidelberg,
Germany, p. 77-94.
[30] Andrews, C. S. 1976. Effect of calcium, pH and nitrogen on the growth and chemical
composition of some tropical and temperate pasture legumes. I. Nodulation and
growth. Australian Journal of Agricultural Research 27: 611-623.
[31] Angelini, J., Castro, S. & Fabra, A. 2003. Alterations in root colonization and nodC
gene induction in the peanut-rhizobia interaction under acidic conditions. Plant
Physiology and Biochemistry 41: 289-294.
[32] Angle, J. S. & Chaney, R. L. 1991. Heavy metal effects on soil populations and heavy
metal tolerance of Rhizobium meliloti, nodulation, and growth of alfalfa. Water, Air
and Soil Pollution 57-58: 597-604.
[33] Antheunisse, J. & Arkestein-Dijksman, L. 1979. Rate of drying and the survival of mi‐
croorganisms. Antonie Van Leeuwenhoek 45: 177-184.
[34] Antheunisse, J., de Bruijn-Tol, J. W. & VanderPol-VanSoest, M. E. 1981. Survival of
microorganisms after drying and storage. Antonie Van Leeuwenhoek 47: 539-545.
[35] Aranjuelo, I., Yrigoyen, J. J. & Sanchez-Diaz, M. 2007. Effect of elevated temperature
and water availability on CO2 exchange and nitrogen fixation of nodulated alfalfa
plants. Environmental and Experimental Botany 59: 99-108.
[36] Arayankoon, T., Schomberg, H. H. & Weaver, R. W. 1990. Nodulation and N2 fixation
of guar at high root temperature. Plant and Soil 126: 209-213.
[37] Arora, N. K., Khare, E., Singh, S. & Maheshwari, D. K. 2010. Effect of Al and heavy
metals on enzymes of nitrogen metabolism of fast and slow growing rhizobia under
explanta conditions. World Journal of Microbiology and Biotechnology 26: 811-816.
[38] Avelar Ferreira, P. A., Lopes, G., Bomfeti, C. A., de Oliveira Longatti, S. M., de Sousa
Soares, C. R., Guimarães Guilherme, L. R. & de Souza Moreira, F. M. 2013. Legumi‐
nous plants nodulated by selected strains of Cupriavidus necator grow in heavy metal
contaminated soils amended with calcium silicate. World Journal of Microbiology
and Biotechnology. DOI 10.1007/s11274-013-1369-2.
163
164
Advances in Biology and Ecology of Nitrogen Fixation
[39] Ayansina, A. D. V. 2009. Pesticide use in agriculture and microorganisms, In: Khan,
M. S., Zaidi, A. & Musarrat, J. (ed.), Microbes in Sustainable Agriculture. Nova Sci‐
ence Publishers, New York, p. 261-284.
[40] Baath, E. 1992. Measurement of heavy metal tolerance of soil bacteria using thymi‐
dine incorporation into bacteria extracted after homogenization-centrifugation. Soil
Biology and Biochemistry 24: 1167-1172.
[41] Baath, E., Diaz-Ravina, M., Frostegard, A. & Campbell, C. D. 1998. Effect of metalrich sludge amendments on the soil microbial community. Applied and Environmen‐
tal Microbiology 64: 238-245.
[42] Badri, M., Soula, S., Huguet, T. & Aouani, M. E. 2010. Symbiotic performance of Med‐
icago ciliaris lines in association with different Sinorhizobium strains. Journal of Eco‐
biotechnology 2: 6-11.
[43] Baldani, J. I. & Weaver, R. W. 1992. Survival of clover rhizobia and their plasmidcured derivatives in soil under heat and drought stress. Soil Biology and Biochemis‐
try 24: 737-742.
[44] Bandyopadhyay, A. K., Jain, V. & Nainawatee, H. S. 1996. Nitrate alters the flavonoid
profile and nodulation in pea (Pisum sativum L.). Biology and Fertility of Soils 21:
189-191.
[45] Barea, J. M. & Azcon-Aguilar, C. 1983. Mycorrhizas and their significance in nodulat‐
ing nitrogen-fixing plants. Advances in Agronomy 36: 1-54.
[46] Becana, M., Aparicio-Rejo, P., Pena, J., Aquirreotea, J. & Sanchez-Diaz, M. 1986. N2
fixation (C2H2-reducing activity) and leghemoglobin content during nitrate and wa‐
ter stress induced senescence of Medicago Sativa root nodules. Journal of Experimen‐
tal Botany 37: 597-605.
[47] Begum, A. A., Leibovitch, S., Migner P. & Zhang, F. 2001. Inoculation of pea (Pisum
sativum L.) by Rhizobium leguminosarum bv. viceae preincubated with naringenin and
hesperetin or application of naringenin and hesperetin directly into soil increased
pea nodulation under short season conditions. Plant and Soil 237: 71-80.
[48] Bellinaso, M. L., Greer, C. W., Peralba, M. C., Henriques, J. A. & Gaylarde, C. C. 2003.
Biodegradation of the herbicide trifluralin by bacteria isolated from soil. FEMS Mi‐
crobiology Ecology 43: 191-194.
[49] Ben Salah, I., Albacete, A., Martinez Andujar, C., Haouala, R., Labidi, N., Zribi, F.,
Martinez, V., Perez-Alfocea, F. & Abdelly, C. 2009. Response of nitrogen fixation in
relation to nodule carbohydrate metabolism in Medicago ciliaris lines subjected to salt
stress. Journal of Plant Physiology 166 (5): 477-488.
[50] Benhizia, Y., Benhizia, H., Benguedouar, A., Muresu, R., Giacomini, A. & Squartini,
A. 2004. Gamma proteobacteria can nodulate legumes of the genus Hedysarum. Sys‐
tematic and Applied Microbiology 27: 462-468.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[51] Bernard, T., Pocard, J. A., Perroud, B. & Le Rudulier, D. 1986. Variations in the re‐
sponse of salt-stressed Rhizobium strains to betaines. Archives of Microbiology 143:
359-364.
[52] Bertholet, J. & Clark, W. 1985. Effect of Trifluralin and Metribuzin on faba bean
growth, development and symbiotic nitrogen fixation. Canadian Journal of Plant Sci‐
ence 65: 9-20.
[53] Bewley, J .B. 1981. Protein synthesis. In: Paleg, L. G. and Aspinall, D. (ed.), The Physi‐
ology and Biochemistry of Drought Resistance in Plants. Academic Press, New York,
p. 261-282.
[54] Bhagwat, A. A., Gross, K. C., Tully, R. E. & Keister, D. L. 1996. Betaglucan synthesis
in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta(1leads to 6) linkages. Journal of Bacteriology 178: 4635-4642.
[55] Bhandal, I. S., Kaur, H. & Bhandal, B. K. 1990. Heavy metal inhibition of in vitro Rhi‐
zobium, growth, nodulation and nitrogen-fixation in Vigna radiata. Phyton 30: 57-63.
[56] Billi, D. & Potts, M. 2002. Life and death of dried prokaryotes. Research in Microbiol‐
ogy 153: 7-12.
[57] Boddey, R. M., Sa, J. C., Alves, B. J. & Urquiaga, S. 1997. The contribution of biologi‐
cal nitrogen fixation for sustainable agricultural systems in the tropics. Soil Biology
and Biochemistry 29: 787-799.
[58] Bolaños, L., El-Hamdaoui, A. & Bonilla, I. 2003. Recovery of development and func‐
tionality of nodules and plant growth in saltstressed Pisum sativum – Rhizobium legu‐
minosarum symbiosis by boron and calcium. Journal of Plant Physiology 160:
1493-1497.
[59] Boncompagni, E., Østerås, M., Poggi, M. C. & Le Rudulier, D. 1999. Occurrence of
choline and glycine betaine uptake metabolism in the family Rhizobiaceace and their
in osmoprotection. Applied and Environmental Microbiology 65: 2072-2077.
[60] Borges, A. & Wollum, A. 1981. Effect of cadmium on symbiotic soybean plants. Jour‐
nal of Environmental Quality 10: 216-221.
[61] Botsford, J. L. 1984. Osmoregulation in Rhizobium meliloti: inhibition of growth by
salts. Archives of Microbiology 137:124-127.
[62] Botsford, J. L. & Lewis, T. A. 1990. Osmoregulation in Rhizobium meliloti: production
of glutamic acid in response to osmotic stress. Applied and Environmental Microbi‐
ology 56: 488-494.
[63] Boumahdi, M., Mary, P. & Hornez, J. 1999. Influence of growth phases and desicca‐
tion on the degrees of unsaturation of fatty acids and the survival rates of rhizobia.
Journal of Applied Microbiology 87: 611-619.
165
166
Advances in Biology and Ecology of Nitrogen Fixation
[64] Bouquard, C., Ouazzani, J., Prome, J., Michel-Briand, Y. & Plesiat, P. 1997. Dechlori‐
nation of atrazine by a Rhizobium sp. isolate. Applied and Environmental Microbiolo‐
gy 63 (3): 862-866.
[65] Bowen, G. D. & Kennedy, M. M. 1956. Effect of high soil temperatures on Rhizobium
spp. Queensland Journal of. Agricultural Science 18: 177-196.
[66] Braswell, B. H., Schimel, D. S., Linder, E. & Moore, B. 1997. The response of global
terrestrial ecosystems to interannual temperature variability. Science 238: 870–872.
[67] Braun, A. D. 1997. Microbial Water Stress. Bacteriology Review 40: 803-846
[68] Breedveld, M. W., Zevenhuizen, L. P. T. M. & Zehnder, A. J. B. 1990. Osmotically in‐
duced oligo- and polysaccharide synthesis by Rhizobium meliloti SU-47. Journal of
General Microbiology 136: 2511-2519.
[69] Breedveld, M. W., Dijkema, C., Zevenhuizen, L. & Zehnder, A. J. B. 1993. Response of
intracellular carbohydrates to a NaCl shock in Rhizobium leguminosarum biovar trifolii
TA1 and Rhizobium meliloti SU47. Journal of General Microbiology 139: 3157-3163.
[70] Bremer, E. & Krämer, R. 2000. Coping with osmotic challenges: osmoregulation
through accumulation and release of compatible solutes in bacteria. In: Storz, G. and
Hengge-Aronis, R. (ed.), Bacterial Stress Response, p. 79-97.
[71] Broos, K., Uyttebroek, M., Mertens, J. & Smolders, E. 2004. A survey of symbiotic ni‐
trogen fixation by white clover grown on metal contaminated soils. Soil Biology and
Biochemistry 36: 633-640.
[72] Broughton, W. J., Zhang, F., Perret, X. & Staehelin C. 2003. Signals exchanged be‐
tween legumes and Rhizobium, agricultural uses and perspectives. Plant and Soil 252:
129-137.
[73] Brown, A. D. 1990. Microbial water stress physiology, principles and perspectives.
John Wiley & Sons, Inc., New York.
[74] Bushby, H. V. A. & Marshall, K. C. 1976. Some factors affecting the survival of rootnodule bacteria on desiccation. Soil Biology and Biochemistry 9: 143-147.
[75] Bushby, H. V. A. & Marshall, K. C. 1977. Desiccation induced damage to the cell en‐
velope of root-nodule bacteria. Soil Biology and Biochemistry 9: 149-152.
[76] Callaham, D. A. & Torrey, J. G. 1981. The structural basis for infection of root hairs of
Trifolium repens by Rhizobium. Canadian Journal of Botany 59: 1647-1664.
[77] Ceribasi, I. H. & Yetis, U. 2001. Biosorption of Ni (II) and Pb (II) by Phanaerochaete
chrysosporium from a binary metal system kinetics. Water SA 27: 15-20.
[78] Chalk, P. M., Souza, R. D. F., Urquiaga, S., ALves, B. J. R. & Boddy, R. M. 2006. The
role of arbuscular mycorrhiza in legume symbiotic performance. Soil Biology and Bi‐
ochemistry 38: 2944-2951.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[79] Chao, W. L. & Alexander, M. 1984. Mineral soils as carriers for Rhizobium inoculants.
Applied and Environmental Microbiology 47: 94-97.
[80] Chaudri, A. M., McGrath, S. P., Giller, K. E., Reitz, E. & Suerbeck, D. R. 1993. Enu‐
meration of indigenous Rhizobium leguminosarum biovar trifolii in soils previously
treated with metal contaminated sewage sludge. Soil Biology and Biochemistry 25:
301-309.
[81] Chaudri, A. M., Allain, C. M., Barbosa-Jefferson, V. L., Nicholson, F. A., Chambers, B.
J. & McGrath, S. P. 2000. A study of the impacts of Zn and Cu on two rhizobial spe‐
cies in soils of a long term field experiment. Plant and Soil 22: 167-179.
[82] Chen, H., Richardson, A. E. & Rolfe, B. G. 1993. Studies of the physiological and ge‐
netic basis of acid tolerance in Rhizobium leguminosurum biovar trifolii. Applied and
Environmental Microbiology 59: 1798-1804.
[83] Chen, L. S., Figueredo, A., Villani, H., Michajluk, J. & Hungria, M. 2002. Diversity
and symbiotic effectiveness of rhizobia isolated from field-grown soybean nodules in
Paraguay. Biology and Fertility of Soils 35: 448-457.
[84] Chen, M. & Alexander, M. 1973. Survival of soil bacteria during prolonged desicca‐
tion. Soil Biology and Biochemistry 5: 213-221.
[85] Chen, W. M., Laevens, S., Lee, T. M., Coenye, T., De Vos, P., Mergeay, M. & Van‐
damme, P. 2001. Ralstonia taiwanensis sp nov., isolated from root nodules of Mimosa
species and sputum of a cystic fibrosis patient. International Journal of Systematic
and Evolutionary Microbiology 51: 1729-1735.
[86] Chen, W. M., Wu, C. H., James, E. K. & Chang, J. S. 2008. Metal biosorption capability
of Cupriavidus taiwanensis and its effects on heavy metal removal by nodulated Mimo‐
sa pudica. Journal of Hazardous Materials 151: 364-371.
[87] Chen, W. M., Moulin, L., Bontemps, C., Vandamme, P., Béna, G. & Boivin-Masson, C.
2003. Legume symbiotic nitrogen fixation by beta-Proteobacteria is widespread in na‐
ture. Journal of Bacteriology 185: 7266-7272.
[88] Cheng, Q. 2008. Perspectives in biological nitrogen fixation research. Journal of Inte‐
grative Plant Biology, 50 (7): 784–796
[89] Cheung, K. H. & Gu, J. D. 2007. Mechanism of hexavalent chromium detoxification
by microorganisms and bioremediation application potential: a review. International
Biodeterioration and Biodegradation 59: 8-15.
[90] Chianu, J. N., Vanlauwe, B., Mahasi, J. M., Katungi, E., Aketch, C., Mairura, F.S.,
Chianu, Justina N & Sanginga, N. 2008. Soybean situation and Outlook analysis; the
case of Kenya.TSBF-CIAT, AFNET, p. 68.
167
168
Advances in Biology and Ecology of Nitrogen Fixation
[91] Chien, C. T., Shetty, K., Mortimer, M. & Orser, C. S. 1991. Calcium-induced salt toler‐
ance in Rhizobium leguminosarum biovar viciae strain C1204b. FEMS Microbiology Let‐
ters 83: 219-224.
[92] Chien, C. T., Maundu, J., Cavaness, J., Dandurand, L. M. & Orser, C. S. 1992. Charac‐
terization of salt-tolerant and salt-sensitive mutants of Rhizobium leguminosarum bio‐
var viciae strain C12046. FEMS Microbiology Letters 90: 135-140.
[93] Cooper, J. E. 1988. Nodulation of legumes by rhizobia in acid soils. In: Vancura, V.
and Kunc, F. (ed.), Developments in soil science. Elsevier Science, Amsterdam, p
57-61.
[94] Cordovilla, M. D. P., Berrido, S. L., Ligero, F. & Lluch, C. 1999. Rhizobium strain ef‐
fects on the growth and nitrogen assimilation in Pisum sativum and Vicia faba plant
growth under salt stress. Journal of Plant Physiology 154: 127-131.
[95] Coventry, D. R. & Evans, J. 1989. Symbiotic nitrogen fixation and soil acidity. In: Rob‐
son, A. D. (ed.), Soil Acidity and Plant Growth. Academic Press, Sydney, p. 103-137.
[96] Crockford, A. J., Davis, G. A. & Williams, H. D. 1995. Evidence for cell-dependent
regulation of catalase activity in Rhizobium leguminosarum bv. phaseoli. Microbiology
141: 843-851.
[97] Cunningham, S. D. & Munns, D. N. 1984. The correlation of the exopolysaccharide
production and acid-tolerance in Rhizobium. Soil Science Society of America Journal
48: 1273-1276.
[98] Curley, R. L. & Burton, J. C. 1975. Compatibility of Rhizobium japonicum with chemi‐
cal seed protectants. Agronomy Journal 67: 807-808.
[99] Cytryn, E. D., Sangurdekar, D. P., Streeter, J. G., Franck, W. L., Chang, W., Stacey, G.,
Emerich, D. W., Joshi, T., Xu, D. & Sadowsky, M. J. 2007. Transcriptional and physio‐
logical responses of Bradyrhizobium japonicum to desiccation-induced stress. Journal
of Bacteriology 189: 6751-6762.
[100] Danese, P. N., Pratt, L. A. & Kolter, R. 2000. Exopolysaccharide production is re‐
quired for development of Escherichia coli K-12 biofilm architecture. Journal of Bac‐
teriology 182: 3593-3596.
[101] Danso, S. K. A. 1995. Assessment of biological nitrogen fixation. Fertililizer Research,
42: 33-41.
[102] Dardanelli, M. S., Fernandez de Cordoba, F. J., Espuny, M. R., Rodrıguez Carvajal M.
A., Soria Diaz, M. E., Gil Serrano, A. M., Okon, Y. & Megıas, M. 2008. Effects of Azo‐
spirillum brasilense coinoculated with Rhizobium on Phaseolus vulgaris flavonoids and
Nod factor production under salt stress. Soil Biology and Biochemistry 40: 2713-2721.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[103] Dart, P. J. & Mercer, F. V. 1965. The effect of growth temperature, level of ammonium
nitrate and light intensity on the growth and nodulation of cowpea (Vigna sinensis
Endl. ex. Hassk.). Australian Journal of Agricultural Research 16: 321-345.
[104] Date, R. A. 2000. Inoculated legumes in cropping systems of the tropics. Field Crops
Research 65: 123-136.
[105] Day, D. A. & Copeland, L. 1991. Carbon metabolisms and compartmentation in nitro‐
gen fixing legume nodules. Plant Physiology and Biochemistry 29: 185-201.
[106] de Lira Juniora, M., Limaa, A. S. T., Arruda, J. R. F. & Smith, D. L. 2005. Effect of root
temperature on nodule development of bean, lentil and pea. Soil Biology and Bio‐
chemistry 37: 235-239.
[107] de Lucena, D. K., Pühler, A. & Weidner, S. 2010. The role of sigma factor RpoH1 in
the pH stress response of Sinorhizobium meliloti. BMC Microbiology 10: 265. doi:
10.1186/1471-2180-10-265.
[108] Deaker, R., Roughley, R. J. & Kennedy, I. R. 2004. Legume seed inoculation technolo‐
gy – a review. Soil Biology and Biochemistry 36: 1275-1288.
[109] Dénarié, J., Debelle, F. & Promé, J. C. 1996. Rhizobium lipo-chito-ligosaccharide nodu‐
lation factors: Signalling molecules mediating recognition an morphogenesis. Annual
Review of Biochemistry 65: 503-535.
[110] Dick, R. E. & Quinn, J. P. 1995. Glyphosate-degrading isolates from environmental
samples: occurence and pathways of degradation. Applied Microbiology and Bio‐
technology 43 (3): 545-550.
[111] Dominguez-Fererras, A., Pérez-Arnedo, R., Becker, A., Olivares, J., Soto, M. J. & San‐
juán, J. 2006. Transcriptome profiling reveals the importance of plasmid pSymB for
osmoadaptation of Sinorhizobium meliloti. Journal of Bacteriology 188: 7617-7625.
[112] dos Santos, J. B., Ferreira, E. A., Kasuya, M. C. M., da Silva, A. A. & Procopio, S. O.
2005. Tolerance of Bradyrhizobium strains to glyphosate formulations. Crop Protection
24 (6): 543-547.
[113] Dowling, D. N. & Broughton, W. J. 1986. Competition for nodulation of legumes. An‐
nual Review of Microbiology 40: 131-157.
[114] Drouin, P., Sellami, M., Prévost, D., Fortin, J. & Antoun, H. 2010. Tolerance to agri‐
cultural pesticides of strains belonging to four genera of Rhizobiaceae. Journal of En‐
vironmental Science and Health Part B 45: 780-788.
[115] Dunigan, E. P., Frey, J. P., Allen, L. D., McMahon, A., 1972. Herbicidal effects on nod‐
ulation of Glycine max (L.) Merrill. Agronomy Journal 64: 806-808.
[116] Duzan, H. M., Mabood, F., Zhou, X., Souleimanov, A. & Smith, D. L. 2005. Nod factor
induces soybean resistance to powdery mildew. Plant Physiology and Biochemistry
43: 1022-1030.
169
170
Advances in Biology and Ecology of Nitrogen Fixation
[117] Eaglesham, A. R. J. & Ayanaba, A. 1984. Tropical stress ecology of rhizobia, root nod‐
ulation and legume fixation. In: Subba Rao, N. S. (ed.), Current Developments in Bio‐
logical Nitrogen Fixation. Edward Arnold Ltd, London, UK, p. 1-35.
[118] Eaglesham, A., Seaman, B. & Ahmad, H. 1981. High-temperature tolerant “cowpea”
rhizobia. In: Gibson, A. H. and Newton, W. E. (ed.), Current perspectives in nitrogen
fixation. Elsevier/North-Holland Biomedical Press, Canberra-Amsterdam.
[119] Eberbach, P. L. & Douglas, L. A. 1991. Effect of herbicide residues in a sandy loam on
the growth, nodulation and nitrogenase activity (C2H2/C2H4) of Trifolium subterra‐
neum. Plant and Soil 131: 67-76.
[120] El-enany, A. E. & Abd-Alla, M. H. 1995. Cadmium resistance in Rhizobium-faba bean
symbiosis. Synthesis of cadmium binding proteins. Phyton 35: 45-53.
[121] Elsheikh, E. A. E. & Wood, M. 1989. Response of chickpea and soybean rhizobia to
salt: osmotic and specific ion effects of salts. Soil Biology and Biochemistry 21:
889-895.
[122] Elsheikh, E. A. E. & Wood, M. 1990a. Salt effects on survival and multiplication of
chickpea and soybean rhizobia. Soil Biology and Biochemistry 22: 343-347.
[123] Elsheikh, E. A. E. & Wood, M. 1990b. Rhizobia and bradyrhizobia under salt stress:
possible role of trehalose in osmoregulation. Letters in Applied Microbiology 10:
127-129.
[124] Estévez, J., Soria Díaz, M. E., Fernández de Córdoba, F., Móron, B., Manyan, H., Gil,
A., Thomas-Oates, J., van Brussel, A. A. N., Dardanelli, M. S., Sousa, C. & Megías, M.
2009. Different andnew Nod factors produced by Rhizobium tropici CIAT899 follow‐
ing Na+ stress. FEMS Microbiology Letters 293: 220-231.
[125] Euzéby, J. P. 1997. List of bacterial names with standing in nomenclature: a folder
available on the Internet. International Journal of Systematic Bacteriology 47 (2):
590-592.
[126] FAO 2008. FAOSTAT Accessed on http://faostat.fao.org/default.aspx (accessed on
April 2011).
[127] Ferreira, P. A. A., Bomfeti, C. A., Junior, R. S., Soares, B. L., Soares, C. R. F. S. & Mor‐
eira, F. M. S. 2012. Symbiotic efficiency of Cupriavidus necator strains tolerant to zinc,
cadmium, copper and lead. Pesquisa Agropecuária Brasileira 47: 85-95.
[128] Fierer, N. & Jackson, R. B. 2006. The diversity and biogeography of soil bacterial com‐
munities. Proceedings of the National Academy of Sciences USA 103: 626-631.
[129] Fierer, N., Schimel, J. P. & Holden P. A. 2003. Influence of drying rewetting frequen‐
cy on soil bacterial community structure. Microbial Ecology 45: 63-71.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[130] Firmin, J. L., Wilson, K. E., Rossen, L. & Johnston, A. W. B. 1986. Flavonoid activation
of nodulation genes in Rhizobium reversed by other compounds present in plants.
Nature 324: 90-92.
[131] Firmin, J. L., Wilson, K. E., Carlson, R. W., Davies, A. E. & Downie, J. A. 1993. Resist‐
ance to nodulation of cv. Afghanistan peas is overcome by nodX, which mediates an
O-acetylation of the Rhizobium leguminosarum lipo-oligosaccharide nodulation factor.
Molecular Microbiology 10: 351-360.
[132] Fitter, A. H. & Garbaye, J. 1994. Interactions between mycorrhizal fungi and other
soil organisms. Plant and Soil 159: 123-132.
[133] Foster, J. W. 2000. Microbial responses to acid stress. In: Storz, G. and Hengge-Aro‐
nis, R. (ed.), Bacterial Stress Response. ASM Press, Washington, D. C., p. 99-115.
[134] Foth, H. D. 1990. Fundamentals of soil science. Wiley, New York.
[135] Fougere, F. & Le Rudulier, D. 1990. Uptake of glycine betaine and its analogues by
bacteroids of Rhizobium meliloti. Journal of General Microbiology 136: 157-163.
[136] Fox, J. E., Starcevic, M., Kow, K. Y., Burow, M. E. & McLachlan, J. A. 2001. Nitrogen
fixation: Endocrine disrupters and flavonoid signaling. Nature 413: 128-129.
[137] Fox, J. E., Starcevic, M., Jones, P. E., Burow, M. E. & McLachlan, J. A. 2004. Phytoes‐
trogen signaling and symbiotic gene activation are disrupted by endocrine-disrupt‐
ing chemicals. Environmental Health Perspectives 112: 672-677.
[138] Fox, J. E., Gulledge, J., Engelhaupt, E., Burow, M. E. & McLachlan, J. A. 2007. Pesti‐
cides reduce symbiotic efficiency of nitrogen-fixing rhizobia and host plants. Pro‐
ceedings of the National Academy of Sciences of the United States of America
104(24): 10282-10287.
[139] Frerot, H., Lefebvre, C., Gruber, W., Collin, C., Dos Santos, A. & Escarre, J. 2006. Spe‐
cific interactions between local metallicous plants improve the phyto stabilisation of
mine soils. Plant and Soil 282: 53-65.
[140] Frings, J. F. J. 1976. The Rhizobium-pea symbiosis as affected by high temperatures.
Ph.D. Thesis.Wageningen Agricultural University, Germany.
[141] Fujihara, S. & Yoneyama, T. 1993. Effects of pH and osmotic stress on cellular polya‐
mine contents in the soybean Rhizobia fredii P220 and Bradyrhizobium japonicum
A1017. Applied and Environmental Microbiology 59: 1104-1109.
[142] Fujishige, N. A., Kapadia, N. N., De Hoff, P. L. & Hirsch, A. M. 2006. Investigations
of Rhizobium biofilm formation. FEMS Microbiology Ecology 56: 195-205.
[143] Gadd, G. M. 1992. Metals and microorganisms: a problem of definition. FEMS Micro‐
biology Letters 100: 197-204.
171
172
Advances in Biology and Ecology of Nitrogen Fixation
[144] Gagnon, H. & Ibrahim, R. K. 1998. Aldonic acids: a novel family of nod gene induc‐
ers of Mesorhizobium loti, Rhizobium lupini and Sinorhizobium meliloti. Molecular PlantMicrobe Interactions 11: 988-998.
[145] Galinski, E. A. 1995. Osmoadaptation in bacteria. Advances in Microbial Physiology
37: 273-328.
[146] Georgiev, G. I. & Atkins, C. A. 1993. Effects of salinity on N 2 fixation, nitrogen metab‐
olism and export and diffusive conductance of cowpea nodules. Symbiosis 15:
239-255.
[147] Gerosa-Ramos, M. L., Parsons, R., Sprent, J. I. & James, E. K. 2003. Effect of water
stress on nitrogen fixation and nodule structure of common bean. Pesquisa Agrope‐
cuária Brasileira 38: 339-347.
[148] Gewaily, E. M., Khan, M. F. & Kheder, A. K. 1991. Effect of temperature and calcium
carbonate contents of soil on the survival and growth of rhizobia. In: 13th North
American Symbiotic Nitrogen Fixation Conference. Banff Centre for Conferences,
Canada, p. 50.
[149] Ghassemi, F., Jakeman, A. J. & Nix, H. A. 1995. Salinization of land and water resour‐
ces: human causes, extent, management and case studies, CAB International, p. 526.
[150] Ghittoni, N. E. & Bueno, M. A. 1996. Changes in the cellular content of trehalose in
our peanut rhizobia strains cultured under hypersalinity. Symbiosis 20: 117-127.
[151] Giller, K. E., McGarth, S. P. & Hirsch, P. R. 1989. Absence of nitrogen fixation in clo‐
ver grown on soil subjected to long-term contamination with heavy metals is due to
survival of only ineffective Rhizobium. Soil Biology and Biochemistry 21: 841-848.
[152] Giller, K. E., Witter, E. & McGrath, S. P. 1998. Toxicity of heavy metals to microor‐
ganisms and microbial process in agricultural soils: a review. Soil Biology and Bio‐
chemistry 30: 1389-1414.
[153] Giller, K. E., Beare, M. H., Lavelle, P., Izac, A. M. N. & Swift, M. J. 1997. Agricultural
intensification, soil biodiversity and agroecosystem function. Applied Soil Ecology 6:
3-16.
[154] Glenn, A. R. & Dilworth, M. J. 1994. The life of root nodule bacteria in the acidic un‐
derground. FEMS Microbiology Letters 123: 1-10.
[155] Gloux, K. & Le Rudulier, D. 1989. Transport and catabolism of proline betaine in saltstressed Rhizobium meliloti. Archives of Microbiology 151: 143-148.
[156] Gonzalez-Gonzalez, R., Botsford, J. L. & Lewis, T. 1990. Osmoregulation in Rhizobium
meliloti: Charaterization of enzymes involved in glutamate synthesis. Canadian Jour‐
nal of Microbiology 36: 469-474.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[157] Gouffi, K. & Blanco, C. 2000. Is the accumulation of osmoprotectant the unique mech‐
anism involved in bacterial osmoprotection? International Journal of Food Microbiol‐
ogy 55: 171-174.
[158] Gouffi, K., Bernard, T. & Blanco, C. 2000. Osmoprotection by pipecolic acid in Sino‐
rhizobium meliloti: specific effects of D and L isomers. Applied and Environmental
Microbiology 66: 2358-2364.
[159] Gouffi, K., Pica, N., Pichereau, V. & Blanco, B. 1999. Disaccharides as a new class of
nonaccumulated osmoprotectants for Sinorhizobium meliloti. Applied and Environ‐
mental Microbiology 65: 1491-1500.
[160] Graham, P. H. & Vance, C. P. 2000. Nitrogen fixation in perspective: an overview of
research and extension needs. Field Crops Research 65: 93-106.
[161] Graham, P. H., Ocampo, G., Ruiz, L. D. & Duque, A. 1980. Survival of Rhizobium pha‐
seoli in contact with chemical seed protectants. Agronomy Journal 72: 625-626.
[162] Graham, P. H., Sadowsky, M. J., Keyser, H. H., Barnet, Y. M., Bradley, R. S., Cooper,
J. E., De Ley, D. J., Jarvis, B. D. W., Roslycky, E. B., Stijdom, B. W., & Young, J. P. W.
1991. Proposed minimal standards for the description of new genera and species of
root and Stem nodulating bacteria. International Journal of Systematic Bacteriology,
41: 582-587.
[163] Guasch-Vidal, B., Estévez, J., Dardanelli, M. S., Soria-Diaz, M. E., de Córdoba, F. F.,
Balog, C. I., Manyani, H., Gil-Serrano, A., Thomas-Oates, J., Hensbergen, P. J., Deeld‐
er, A. M., Megías, M. & van Brussel, A. A. 2013. High NaCl concentrations induce the
nod genes of Rhizobium tropici CIAT899 in the absence of flavonoid inducers. Molecu‐
lar Plant-Microbe Interactions 26(4): 451-460.
[164] Guerin, V., Trinchant, J. C. & Rigaud, J. 1990. Nitrogen fixation (C2H2 reduction) by
broad bean (Vicia faba L.) nodules and bacteroids under water-restricted conditions.
Plant Physiology 92: 595-601.
[165] Guerin, V., Pladys, D., Trinchant, J. C. & Rigaud, J. 1991. Proteolysis and nitrogen fix‐
ation in faba-bean Vicia faba nodules under water stress. Physiologia Plantarum 82:
360-366.
[166] Gustafson, A. M., O'Connell, K. P. & Thomashow, M. F. 2002. Regulation of Sinorhi‐
zobium meliloti 1021 rrnA-Reporter gene fusions in response to cold shock. Canadian
Journal of Microbiology 48: 821-830.
[167] Hafeez, F. Y., Shah, N. H., Malik, K. A. 2000. Field evaluation of lentil cultivars inocu‐
lated with R. leguminosarum bv. viciae strains for nitrogen fixation using nitrogen-15
isotope dilution. Biology and Fertility of Soils 31: 65-69.
[168] Han, Y., Zhou, D., Pang, X., Zhang, L., Song, Y., Tong, Z., Bao, J., Dai, E., Wang, J.,
Guo, Z., Zhai, J., Du, Z., Wang, X., Wang, J., Huang, P. & Yang, R. 2005. Comparative
173
174
Advances in Biology and Ecology of Nitrogen Fixation
transcriptome analysis of Yersinia pestis in response to hyperosmotic and high-salini‐
ty stress. Research in Microbiology 156: 403-415.
[169] Hansen, A. P. 1994. Symbiotic N2 fixation of crop legumes: Achievement and per‐
spectives, Hohenheim Tropical Agricultural Series. Margraf Verlag, p. 31-36.
[170] Hatimi, A. 1999. Effect of salinity on the association between root symbionts and Aca‐
cia cyanophylla Lind.: growth and nutrition. Plant and Soil 216: 93-101.
[171] Hayman, D. S. 1986. Mycorrhizae of nitrogen-fixing legumes. MIRCEN Journal 2:
121-145.
[172] Hecker, M. & Völker, U. 1998. Non-specific, general and multiple stress resistance of
growth restricted Bacillus subtilis cells by the expression of the σB Regulon. Molecular
Microbiology 29: 1129-1136.
[173] Hecker, M., Schumann, W. & Völker, U. 1996. Heat-shock and general stress re‐
sponse in Bacillus subtilis. Molecular Microbiology 19: 417-428.
[174] Heckman, J. R., Angel J. S. & Chaney R. L. 1984. Residual effects of sewage sludge on
soybean. II. accumulation of soil and symbiotically fixed nitrogen. Journal of Envi‐
ronmental Quality 16: 118-124.
[175] Hengge-Aronis, R. 1999. Interplay of global regulators and cell physiology in the
general stress response of Escherichia coli. Current Opinions in Microbiology 2:
148-152.
[176] Herman P. L., Behrens, M., Chakraborty, S., Crastil, B. M., Barycki, J. & Weeks, D. P.
2005. A three component dicamba O-demethylase from Pseudomonas maltiphilia strain
DI-6: Gene isolation, characterization and heterologous expression. Journal of Biolog‐
ical Chemistry 280: 24759-24767.
[177] Hernandez-Armenta, R., Wien, H. C. & Eaglesham, A. R. J. 1989. Maximum tempera‐
ture for nitrogen fixation in common bean. Crop Science 29: 1260-1265.
[178] Hernandez-Jimenez, M. J., Lucas, M. M. & de Felipe, M. R. 2002. Antioxidant defense
and damage in senescing lupin nodules. Plant Physiology and Biochemistry 40:
645-657.
[179] Hirsch, A.M. 2010. How rhizobia survive in the absence of a legume host, a stressful
world indeed. In Symbioses and Stress: Joint Ventures in Biology, Cellular Origin,
Life in Extreme Habitats and Astrobiology, Seckbach, J. and Gruber, M. (ed.) Spring‐
er Science and Business Media, p. 375-391.
[180] Hirsch, P. R., Jones, M. J., McGrath, S. P. & Giller, K. E. 1993. Heavy metals from past
applications of sewage sludge decrease the genetic diversity of Rhizobium leguminosa‐
rum biovar trifolii populations. Soil Biology and Biochemistry 25: 1485-1490.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[181] Hoglund, J. H. & Brock, J. L. 1987. Nitrogen fixation in managed grasslands. In: Nay‐
don, R. W. (ed.), Managed Grasslands: Analytical studies.Ecosystems of the World
17B. Elsevier, Amsterdam, p. 187-196.
[182] Howieson, J. G., Robson, A. D. & Abbot, L. K. 1992. Acid-tolerant species of Medicago
produce root exudates at low pH which induce the expression of nodulation genes in
Rhizobium meliloti. Australian Journal of Plant Physiology 19: 287-296.
[183] Hsiao, C. T. 1973. Plant responses to water stress. Annual Review of Plant Physiology
24: 519-570.
[184] Hua, S. S. T., Tsai, V. Y., Lichens, G. M. & Noma, A. T. 1982. Accumulation of amino
acid in Rhizobium sp. strain WR1001 in response to sodium chloride salinity. Applied
and Environmental Microbiology 44: 135-140.
[185] Huang, C. Y., Bazzaz, F. A. & Vanderhoef, L. N. 1974. The inhibition of soybean me‐
tabolism by cadmium and lead. Plant Physiology 53: 308-312.
[186] Huang, C. Y., Boyer, J. S. & Vanderhoef, L. N. 1975. Limitation of acetylene reduction
(nitrogen fixation) by photosynthesis in soybean having low water potentials. Plant
Physiology 56: 228-232.
[187] Hungria, M. & Franco, A. A. 1993. Effects of high temperature on nodulation and ni‐
trogen fixation by Phaseolus vulgaris L. Plant and Soil 149: 95-102.
[188] Hungria, M. & Phillips, D. A. 1993. Effects of a seed color mutation on rhizobial nodgene-inducing flavonoids and nodulation in common bean. Molecular Plant-Microbe
Interactions 6: 418-422.
[189] Hungria, M. & Stacey, G. 1997. Molecular signals exchanged between host plants and
rhizobia: Basic aspects and potential application in agriculture. Soil Biology and Bio‐
chemistry 29: 819-830.
[190] Hungria, M. & Vargas, M. A. T. 2000. Environmental factors affecting N2 fixation in
grain legumes in the tropics, with an emphasis on Brazil. Field Crops Research 65:
151-164.
[191] Irigoyen, J. J., Emerich, D. W. & Sanchez-Diaz, M. 1992. Water stress induced changes
in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago
sativa) plants. Physiologia Plantarum 84: 55-60.
[192] Ishaq, Y. Z., Zouhdi, L. I., El Haddad, M. E., Moawad, H., Abdelaziz, R. A. 1989. Pop‐
ulation dynamics of introduced R. trifolii strains in the rhizosphere of Egyptian clo‐
ver in relation to salt stress and pattern of competition. In: Abstracts of the fifth
International Symposium on Microbial Ecology, Kyoto, Japan.
[193] Jiang, W., Hou, Y. & Inouye, M. 1997. CspA, the major cold-shock protein of Escheri‐
chia coli, is an RNA chaperone. Journal of Bacteriology 272: 196-202.
175
176
Advances in Biology and Ecology of Nitrogen Fixation
[194] Johnsen, K., Jacobsen, C. S., Torsvik, V. & Sorensen, J. 2001. Pesticide effects on bacte‐
rial diversity in agricultural soils-a review. Biology and Fertility of Soils 33: 443-453.
[195] Johnson, A. C. & Wood, M. 1990. DNA as a possible site of action of aluminium in
Rhizobium sp. Applied and Environmental Microbiology 56: 3629-3633.
[196] Jones, F. R. & Tisdale, W. B. 1921. Effect of soil temperature upon the development of
nodules on the roots of certain legumes. Journal of Agricultural Research 22: 17-37.
[197] Jurcisek, J. A. & Bakaletz, L. O. 2007. Biofilms formed by nontypeable Haemophilus in‐
fluenzae in vivo contain both double-stranded DNA and type IV pilin protein. Journal
of Bacteriology 189: 3868-3875.
[198] Karanja, N. K. & Wood, M. 1988. Selecting Rhizobium phaseoli strains for use with
beans (Phaseolus vulgaris L.) in Kenya: Tolerance of high temperature and antibiotic
resistance. Plant and Soil 112: 15-22.
[199] Kassem, M., Capellano, A. & Gounot, A. M. 1985. Effets du chlorure de sodium sur la
croissance in vitro, l'infectivité et l'efficience de Rhizobium meliloti. MIRCEN Journal
1: 63-73.
[200] Kaur, C., Maini, P. & Shukla, N. P. 2007. Effect of captan and carbendazim fungicides
on nodulation and biological nitrogen fixation in soybean. Asian Journal of Experi‐
mental Sciences 21 (2): 385-388.
[201] Khan, M. & Scullion, J. 2002. Effects of metal (Cd, Cu, Ni, Pb or Zn) enrichment of
sewage-sludge on soil micro-organisms and their activities. Applied Soil Ecology 20:
145-155.
[202] Khan, M. S., Zaidi, A., Wani, P. A. & Oves, M. 2009. Role of plant growth promoting
rhizobacteria in the remediation of metal contaminated soils. Environmental Chemis‐
try Letters 7: 1-19.
[203] Kiss, E., Huguet, T., Poinsot, V. & Batut, J. 2004. The typA is required for stress adap‐
tation as well as for symbiosis of Sinorhizobium meliloti 1021 with certain Medicago
truncatula Lines. Molecular Plant-microbe Interactions 17: 235-244.
[204] Kluson, A. R., Kenworthy, W. J. & Weber, D. F. 1986. Soil temperature effects on
competitiveness and growth of Rhizobium japonicum and on Rhizobium-induced chlo‐
rosis of soybean. Plant and Soil 95: 201-207.
[205] Kosanke, J. W., Osburn, R. M., Shuppe, G. I. & Smith, R. S. 1991. Slow rehydration
improves the recovery of dried bacterial populations. Canadian Journal of Microbiol‐
ogy 38: 520-525.
[206] Kosslak, R. M., Bookland, R., Barkei, J., Paaren, H. & Appelbaum, E. R. 1987. Induc‐
tion of Bradyrhizobium japonicum common nod genes by isoflavones isolated from
Glycine max. Proceedings of the Natural Academy of Science, USA 84: 7428-7432.
[207] Kozik, A., Heidstra, R., Horvath, B., Kulikova, O., Tikhonovich, I., Ellis, T. H. N., van
Kammen, A., Lie, T. A. & Bisseling, T. 1995. Pea lines carrying sym1 or sym2 can be
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
nodulated by Rhizobium strains containing nodX; sym1 and sym2 are allelic. Plant
Science 108: 41-49.
[208] Krujatz, F., Haarstrick, A., Nortemann, B. & Greis, T. 2012. Assessing the toxic effects
of nickel, cadmium and EDTA on growth of the plant growth-promoting rhizobacte‐
rium Pseudomonas brassicacearum. Water, Air and Soil Pollution 223 (3): 1281-1293.
[209] Kumar, S. 1981. Effect of simazine and prometryne on the growth and nodulation of
chickpea (Cicer arietinum L.). Journal of Agricultural Science 97: 663-671.
[210] Kumar, S., Mukerji, K. G. & Lal, R. 1996. Molecular aspects of pesticide degradation
by microorganisms. Critical Reviews in Microbiology 22: 1-26.
[211] Kurchak, O. N., Provorov, N. A. & Simarov, B. V. 2001. Plasmid pSym1-32 of Rhizobi‐
um leguminosarum bv. viceae controlling nitrogen fixation activity, effectiveness of
symbiosis, competitiveness and acid tolerance. Russian Journal of Genetics 37:
1025-1031.
[212] Kust, C. A. & Struckmeyer, B. E. 1971. Effects of Trifluralin on the growth, nodula‐
tion and anatomy of soybean. Weed Science 19: 147-152.
[213] Lakzian, A., Murphy, P., Turner, A., Beynon, J. L. & Giller, K. E. 2002. Rhizobium legu‐
minosarum bv. viciae populations in soils with increasing heavy metal contamination:
abundance, plasmid profiles, diversity and metal tolerance. Soil Biology and Bio‐
chemistry 34: 519-529.
[214] Lanyon, W. E. 1995. Eastern meadowlark (Sturnella magna). The birds of North Amer‐
ica, number 160. The American Ornithologists' Union, Washington D.C., USA, and
The Academy of Natural Sciences, Philadelphia, Pennsylvania, USA.
[215] Laranjo, M. & Oliveira, S. 2011. Tolerance of Mesorhizobium type strains to different
environmental stresses. Antonie Van Leeuwenhoek 99: 651-662.
[216] Lentze, N., Aquilina, J. A., Lindbauer, M., Robinson, C. V. & Narberhaus, F. 2004.
Temperature and concentration-controlled dynamics of rhizobial small heat shock
proteins. European Journal of Biochemistry 271: 2494-2503.
[217] Lie, T. A. 1984. Host genes in Pisum sativum L. conferring resistance to European Rhi‐
zobium leguminosarum strains. Plant and Soil 82: 415-425.
[218] Lindström, K., Terefewrok, Z., Suominen, L. & Lortet, G. 2002. Signalling and devel‐
opment of Rhizobium-legume symbioses. Biology and Environment: Proceedings of
the Royal Irish Academy 1: 61-64.
[219] Liu, C. M., McLean, P. A., Sookdeo, C. C. & Cannon, F. C. 1991. Degradation of the
herbicide glyphosate by members of the family Rhizobiaceae. Applied and Environ‐
mental Microbiology 57 (6): 1799-1804.
177
178
Advances in Biology and Ecology of Nitrogen Fixation
[220] Lloret, J., Wulff, B. B. H., Rubio, J. M., Downie, J. A., Bonilla, I. & Rivilla, R. 1998. Exo‐
polysaccharide II production is regulated by salt in the halotolerant strain Rhizobium
meliloti EFB1. Applied and Environmental Microbiology 64: 1024-1028.
[221] Long, S. R. 1989. Rhizobium genetics. Annual Review of Genetics 23: 483-506.
[222] Long, S. R. 2001. Genes and signals in the Rhizobium-legume symbiosis. Plant Physi‐
ology 125: 69-72.
[223] Lynch, D. H. & Smith, D. L. 1994. The effect of low root zone temperature stress on
two soybean (Glycine max) genotypes when combined with Bradyrhizobium strains of
varying geographic origin. Physiologia Plantarum 90: 105-113.
[224] Madkour, M. A., Smith, L. T. & Smith, G. M. 1990. Preferential osmolyte accumula‐
tion: a mechanism of osmotic stress adaptation in diazotrophic bacteria. Applied and
Environmental Microbiology 56: 2876-2881.
[225] Mahieu, S., Frerot, H., Vidal, C., Galiana, A., Heulin, K. & Maure, L. 2011. Anthyllis
vulneraria / Mesorhizobium metallidurans, an efficient symbiotic nitrogen fixing associa‐
tion able to grow in mine tailings highly contaminated by Zn, Pb and Cd. Plant and
Soil 342: 405-417.
[226] Mahmoud, A. L. E. & Omar, S. A. 1995. Growth, cell wall-degrading enzymes and
aflatoxin production by lemon-rotting fungi in relation to insecticide application. Mi‐
crobiological Research 150: 195-200.
[227] Mallik, M. A. B. & Tesfai, K. 1983. Compatibility of Rhizobium japonicum with com‐
mercial pesticides in vitro. Bulletin of Environmental Contamination and Toxicology
31: 432-437.
[228] Mandal, S. M., Gouri, S. S., De, D. Das, B. K. Mondal, K. C., Bikas R & Pati, B. R. 2011.
Effect of arsenic on nodulation and nitrogen fixation of blackgram (Vigna mungo).In‐
dian Journal of Microbiology 51(1): 44-47.
[229] Marcar, N. E., Dart, P. & Sweeney, C. 1991. Effect of root-zone salinity on growth and
chemical composition of Acacia ampliceps B. R. Maslin, A. auriculiformis A. Cunn ex
Benth and A. mangium Willd at two nitrogen levels. New Phytologist 119: 567-573.
[230] Martin, R. B. 1988. The chemistry of aluminium as related to biology and medicine.
Clinical Chemistry 23: 1797-1806.
[231] Martin, R. B. 1991. Aluminium in biological systems. In: Nicolini, M., Zatta, P. F. and
Corain, B. (ed.), Aluminium in Chemistry, Biology and Medicine. Aseries of advan‐
ces. Raven Press, New York, p 3-20.
[232] Martinez-Romero, E. 2003. Diversity of Rhizobium-Phaseolus vulgaris symbiosis: over‐
view and perspectives. Plant and Soil 252: 11-23.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[233] Mary, P., Ochin, D. & Tailliez, R. 1985. Rates of drying and survival of Rhizobium me‐
liloti strains during storage at different relative humidities. Applied and Environ‐
mental Microbiology 50: 207-211.
[234] Mary, P., Ochin, D. & Tailliez, R. 1986. Growth status of rhizobia in relation to their
tolerance to low water activities and desiccation stress. Soil Biology and Biochemistry
18: 179-184.
[235] Mary, P., Dupuy, C., Biremon, C. D., Defives, C. & Tailliez, R. 1994. Differences
among Rhizobium meliloti and Bradyrhizobium japonicum strains in tolerance to desic‐
cation and storage at different relative humidities. Soil Biology and Biochemistry 26:
1125-1132.
[236] Matsukawa, M. & Greenberg E. P. 2004. Putative exopolysaccharide synthesis genes
influence Pseudomonas aeruginosa biofilm development. Journal of Bacteriology 186:
4449-4456.
[237] Mattimore, V. & Battista, J. R. 1996. Radioresistance of Deinococcus radiodurans: func‐
tions necessary to survive ionizing radiation are also necessary to survive prolonged
desiccation. Journal of Bacteriology 178: 633-637.
[238] McGrath, S. P. & Lane, P. W. 1989. An explanation for the apparent losses of metals
in a long-term field experiment with sewage sludge. Environmental Pollution 60:
235-256.
[239] McGrath, S. P., Hirsch, P. R. & Gillar, K. E. 1988. Effects of heavy metals contamina‐
tion on the genetics of nitrogen fixing populations of Rhizobium legumonosarum bv.
trifolii. In: Orio, A. A. (ed.), Proceedings of the International Conference of Environ‐
mental Contamination. Venice, Italy, p. 164-166.
[240] Mcilveen, W. & Cole, H. 1974. Influence of heavy metals on nodulation of red clover.
Phytopathology 64: 583-589.
[241] Mcintyre, J. J., Davies, H., Hore, T. A., Miller, S. H., Dufour, J. P.& Ronson, C. W.
2007. Trehalose biosynthesis in Rhizobium leguminosarum bv. trifolii and its role in des‐
iccation tolerance. Applied and Environmental Microbiology 73: 3984-3992.
[242] McKay, I. A. & Djordjevic, M. A. 1993. Production and excretion of nod metabolites
by Rhizobium leguminosarum bv. trifolii are disrupted by the same environmental fac‐
tors that reduce nodulation in the field. Applied and Environmental Microbiology
59: 3385-3392.
[243] McLean, E. O. 1976. Chemistry of soil aluminium. Communications in Soil Science
and Plant Analysis 7: 619-636.
[244] Meghvansi, M. K. & Mahna, S. K. 2009. Evaluating the symbiotic potential of Glomus
intraradices and Bradyrhizobium japonicum in Vertisol with two soybean cultivars.
American-Eurasian Journal of Agronomy 2(1): 21-25.
179
180
Advances in Biology and Ecology of Nitrogen Fixation
[245] Mergaert, P., van Montagu, M. & Holsters, M. 1997. Molecular mechanisms of Nod
factor diversity. Molecular Microbiology 25: 811-817.
[246] Mergeay, M., Monchy, S., Vallaeys, T., Auquier, V., Benotmane, A., Bertin, P., Tagha‐
vi, S., Dunn, J., Van der Lelie, D. & Wattiez, R. 2003. Ralstonia metalllidurans, a bacteri‐
um specifically adapted to toxic metals: towards a catalogue of metal-responsive
genes. FEMS Microbiology Reviews 27: 385-410.
[247] Meyer, D. R. & Anderson, A. J. 1959. Temperature and symbiotic nitrogen fixation.
Nature 183: 161.
[248] Michiels, J., Verreth, C. & Vanderleyden, J. 1994. Effects of temperature stress on
bean-nodulating Rhizobium strains. Applied and Environmental Microbiology 60:
1206-1212.
[249] Miettinen, P. & Echegoyen, P. E. 1996. The effect of two pesticides (Vitavax-300 and
Gaucho) on rhizobia and on the nodulation of four legumes. Agriculture Food and
Science Finland 5: 203-207.
[250] Miller, K. J. & Wood, J. M. 1996. Osmoadaptation by rhizosphere bacteria. Annual
Reviews of Microbiology 50: 101-136.
[251] Miransari, M. & Smith, D. L. 2007. Overcoming the stressful effects of salinity and
acidity on soybean [Glycine max (L.) Merr.] nodulation and yields using signal mole‐
cule genistein under field conditions. Journal of Plant Nutrition 30: 1967–1992.
[252] Miransari, M. & Smith, D.L. 2008. Using signal molecule genistein to alleviate the
stress of suboptimal root zone temperature on soybean-Bradyrhizobium symbiosis
under different soil textures. Journal of Plant Interactions 3: 287–295.
[253] Miransari, M. & Smith, D. L. 2009. Alleviating salt stress on soybean [Glycine max (L.)
Merr] – Bradyrhizobium japonicum symbiosis using signal molecule genistein. Europe‐
an Journal of Soil Biology 45: 146-152.
[254] Mohammadi, K., Sohrabi, Y., Heidari, G., Khalesro, S. & Majidi, M. 2012. Effective
factors on biological nitrogen fixation. African Journal of Agricultural Research 7(12):
1782-1788.
[255] Morón, B., Soria-Diaz, M. E., Ault, J., Verrolos, G., Noreen, S., Rodriquez-Navarro, D.
N., Gil-Serrano, A., Thomas-Oates, J., Megias, M. & Sousa, C. 2005. Low pH changes
the profile of nodulation factors produced by Rhizobium tropici CIAT899. Chemistry
and Biochemistry 12: 1029-1040.
[256] Moulin, L., Munive, A., Dreyfus, B. & Boivin-Masson, C. 2001. Nodulation of le‐
gumes by members of the beta-subclass of Proteobacteria. Nature 411: 948-950.
[257] Mpepereki, S., Makonese, F. & Wollum, A. G. 1997. Physiological characterization of
indigenous rhizobia nodulating Vigna unguiculata in Zimbabwean soils. Symbiosis
22: 275-292.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[258] Muglia, C. I., Grasso, D. H. & Aguilar, O. M. 2007. Rhizobium tropici response to acidi‐
ty involves activation of glutathione synthesis. Microbiology 153: 1286-1296.
[259] Münchbach, M., Nocker, A. & Narberhaus, F. 1999. Multiple small heat shock pro‐
teins in rhizobia. Journal of Bacteriology 181: 83- 90.
[260] Munevar, F. & Wollum, A. G. 1981. Effect of high root temperature and Rhizobium
strain on nodulation, nitrogen fixation, and growth of soybeans. Soil Science Society
of America Journal 45: 1113-1120.
[261] Munns, D. N. 1986. Acid soils tolerance in legumes and rhizobia. Advances In Plant
Nutrition 2: 63-91.
[262] Munns, R. & Termaat, A. 1986. Whole-plant responses to salinity. Australian Journal
of Plant Physiology 13: 143-60.
[263] Munns, R. & Tester, M. 2008. Mechanisms of salinity tolerance. Annual Review of
Plant Biology 59: 651-681.
[264] Naeem, F., Malik, K. A. & Hafeez, F. Y. 2008. Pisum sativum–Rhizobium interactions
under different environmental stresses. Pakistan Journal of Botany 40: 2601-2612.
[265] Nandal, K., Sehrawat, A. R., Yadav, A. S., Vashishat, R. K. & Boora, K. S. 2005. High
temperature-induced changes in exopolysaccharides, lipopolysaccharides and pro‐
tein profile of heat-resistant mutants of Rhizobium sp. (Cajanus). Microbiological Re‐
search 160: 367-373.
[266] Nandasena, K. G., O'Hara, G. W., Tiwari, R. P., Willems, A. & Howieson, J. G. 2009.
Mesorhizobium australicum sp. nov. and Mesorhizobium opportunistum sp. nov., isolated
from Biserrula pelecinus L. in Australia. International Journal of Systematic and Evolu‐
tionary Microbiology 59: 2140-2147.
[267] Naya, L., Ladrera, R., Ramos, J., Gonzalez, E., Arrese-Igor, C., Minchin, F. R. & Beca‐
na, M. 2007. The response of carbon metabolism and antioxidant defenses of alfalfa
nodules to drought stress and to the subsequent recovery of plants. Plant Physiology
144: 1104-1114.
[268] Netzer, W. J. & Hartl, F. U. 1998. Protein folding in the cytosol: chaperonin-depend‐
ant and -Independent mechanisms. Trends in Biochemical Science 23: 68-73.
[269] Nishiwaki, T., Sato, T., Yashima, H., Ikarashi, T., Ohyama, T., Harper, J.E., Akao, S.,
Kouchi, H. 1997. Changes in concentration of leghemoglobin components in hyper‐
nodulation mutants of soybean, Soil Science and Plant Nutrition, 43: 1091-1096.
[270] Nocker, A., Krstulovic, N. P., Perret, X. & Narberhaus, F. 2001. ROSE elements occur
in disparate rhizobia and are funtionally interchangeable between species. Archives
of Microbiology 176: 44-51.
181
182
Advances in Biology and Ecology of Nitrogen Fixation
[271] O’Connell, K., Gustafson, A. M., Lehmann, M. D. & Thomashow, M. F. 2000. Identifi‐
cation of cold shock gene loci in Sinorhizobium meliloti by using a luxAB reporter
transposon. Applied and Environmental Microbiology 66: 401-405.
[272] Ogutcu, H., Algur, O. F., Elkoca, E. & Kantar, F. 2008. The determination of symbiotic
effectiveness of Rhizobium strains isolated from wild chickpeas collected from high
altitudes in erzurum. Turkish Journal of Agriculture and Forestry 32: 241-248.
[273] Ohyama, T. & Kumazawa, K. 1981. Nitrogen assimilation in soybean nodules V. Pos‐
sible pathway of allantoin synthesis in soybean nodules. Soil Science and Plant Nu‐
trition 27: 111-114.
[274] Ohyama, T., Ohtake, N., Sueyoshi, K., Tewari, K., Takahashi, Y., Ito, S., Nishiwaki, T.,
Nagumo, Y., Ishii, S. & Sato, T. 2009. Nitrogen fixation and metabolism in soybean
plants, Nova Science Publishers, Inc., New York.
[275] Ohyama, T., Fujikake, H., Yashima, H., Tanabata, S., Ishikawa, S., Sato, T., Nishiwaki,
T., Ohtake, N., Sueyoshi, K., Ishii, S. & Fujimaki, S. 2011. Effect of nitrate on nodula‐
tion and nitrogen fixation of soybean. In: El-Shemy, H. (ed.), Soybean Physiology
and Biochemistry. InTech, Shanghai, China. DOI: 10.5772/17992.
[276] Oldroyd, G. E. D. 2001. Dissecting symbiosis: developments in Nod factor signal
transduction. Annals of Botany (London) 87: 709-718.
[277] Oldroyd, G. E. D., Engstrom, E. M. & Long, S. R. 2001. Ethylene inhibits the Nod fac‐
tor signal transduction pathway of Medicago truncatula. The Plant Cell 13: 1835-1849.
[278] Olsthoorn, M. M. A., Stokvis, E., Haverkamp, J., Spaink, H. P. & Thomas-Oates, J. E.
2000. Growth temperature regulation of host specific modifications of rhizobial lipochitin oligosaccharides: the function of nodX is temperature regulated. Molecular
Plant-Microbe Interactions 13: 808-820.
[279] Ortiz-Hernández, M. L. & Sánchez-Salinas, E. 2010. Biodegradation of the organo‐
phosphate pesticide tetrachlorvinphos by bacteria isolated from agricultural soils in
México. Revista internacional de contaminación ambiental 26: 27-38.
[280] Ovtsyna, A. O., Rademaker, G. J, Esser, E., Weinman, J., Rolfe, B. G., Tikhonovich, I.
A., Lugtenberg, B. J. J., Thomas-Oates, J. E. & Spaink, H. P. 1999. Comparison of char‐
acteristics of the nodX genes from various Rhizobium leguminosarum strains. Molecu‐
lar Plant-Microbe Interactions 12: 252-258.
[281] Paivoke, A. 1983a. The long term effects of zinc on the growth and development,
chlorophyll content and nitrogen fixation of the garden pea (Pisum sativum cv Dipple
May). Annales Botanici Fennici 20: 205-213.
[282] Paivoke, A. 1983b. The long term effects of lead and arsenate on the growth and de‐
velopment, chlorophyll content and nitrogen fixation of the garden pea (Pisum sati‐
vum cv. Dipple May). Annales Botanici Fennici 20: 297-306.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[283] Pan, B. & Smith, O. L. 1998. Genistein and daidzein concentrations and contents in
seedling roots of three soybean cultivars grown under three foot zone temperatures.
Journal of Agronomy and Crop Science 180: 77-82.
[284] Pankhurst, C. E. & Sprent, J. I. 1976. Effects of temperature and oxygen tension on the
nitrogenase and respiratory activities of turgid and water-stressed soybeans and
French bean root nodules. Journal of Experimental Botany 27: 1-9.
[285] Panoff, J. M., Corroler, D., Thammavongs, B. & Boutibonnes, P. 1997. Differentiation
between cold shock proteins and cold acclimation proteins in a mesophilic Grampositive bacterium, Enterococcus faecalis JH2-2. Journal of Bacteriology 179: 4451-4454.
[286] Pan-UK 2003. Current Pesticide Spectrum, Global use and Major Concerns.
[287] Paudyal, S. P., Aryal, R. R., Chauhan, S. V. S. & Maheshwari, D. K. 2007. Effect of
heavy metals on growth of Rhizobium strains and symbiotic efficiency of two species
of tropical legumes. Scientific World 5: 27-32.
[288] Paul, M. J. & Cockburn, W. 1989. Pinitol, a compatible solute in Mesembryanthemum
crvstallinum L. Journal of Experimental Botany 40: 1093-1098.
[289] Pawlak-Sprada, S., Arasimowicz-Jelonek, M., Podgórska, M. & Deckert, J. 2011a. Ac‐
tivation of phenylpropanoid pathway in legume plants expose to heavy metals: Part
I. Effects of cadmium and lead on phenylalanine ammonia-lyase gene expression, en‐
zyme activity and lignin content. Acta Biochimica Polonica 58: 211-216.
[290] Pawlak-Sprada, S., Stobiecki, M. & Deckert, J. 2011b. Activation of phenylpropanoid
pathway in legume plants exposed to heavy metals. Part ii. Profiling of isoflavonoids
and their glycoconjugates induced in roots of lupine (Lupinus luteus) seedlings treat‐
ed with cadmium and lead. Acta Biochimica Polonica 58: 217-223.
[291] Pena-Cabrales, J. J. & Castellanos, J. Z. 1993. Effect of water stress on N2 fixation and
grain yield of Phaseolus vulgaris. Plant and Soil 152: 151-155.
[292] Pereira, S. I. A., Lima, A. I. G. & Figueira, E. M. A. P. 2006. Heavy metal toxicity in
Rhizobium leguminosarum biovar viciae isolated from soils subjected to different sour‐
ces of heavy metal contamination: effect on protein expression. Applied Soil Ecology
33: 286-293.
[293] Perret, X., Staehelin, C. & Broughton, W. 2000. Molecular basis of symbiotic promis‐
cuity. Microbiology and Molecular Biology Reviews 64: 180-201.
[294] Phadtare, S., Yamanaka, K. & Inouye, M. 2000. The Cold Shock Response. In: Storz,
G. and Hengge-Aronis, R. (ed.), Bacterial stress response. ASM Press, Washington,
D.C., p. 33-45.
[295] Phillips, D. A. 1999. Using rhizobia in the 21st century. Symbiosis 27: 83-102.
[296] Phillips, D. A. & Torrey, J. G. 1973. Research in grain legume improvement. Span 16:
9-11.
183
184
Advances in Biology and Ecology of Nitrogen Fixation
[297] Piha, M. I. & Munnus, D. N. 1987. Sensitivity of the common bean (Phaseolus vulgaris
L.) symbiosis to high soil temperature. Plant and Soil 98: 183-194.
[298] Pladys, D. & Rigaud, J. 1985. Senescence in French-bean nodules: occurrence of dif‐
ferent proteolytic activities. Physiologia Plantarum 63 (1): 43-48.
[299] Poolman, B., Blount, P., Folgering, J. H. A., Friesen, R. H. E., Moe, P. C. & van der
Heide, T. 2002. How do Membrane Proteins Sense Water Stress? Molecular Microbi‐
ology 44: 899-902.
[300] Porcel, R., Barea, J. M. & Ruiz-Lozano, J. M. 2003. Antioxidant activities in mycorrhi‐
zal soybean plants under drought stress and their possible relationship to the process
of nodule senescence. New Phytologist 157: 135-143.
[301] Postgate, J. R. 1982. The fundamentals of nitrogen fixation. Cambridge University
Press, Cambridge, United Kingdom.
[302] Potts, M. 1994. Desiccation tolerance of prokaryotes. Microbiology Reviews 58:
755-805.
[303] Pueppke, G. S. & Broughton, W. J. 1999. Rhizobium sp. strain NGR234 and R. fredii
USDA257 share exceptionally broad, nested host ranges. Molecular Plant-Microbe In‐
teractions 12: 293-318.
[304] Purwantari, N. D., Date, R. A. & Dart, P. J. 1995. Nodulation and N2 by Calliandra cal‐
othyrsus and Sesbania sesban grown at different root temperatures. Soil Biology and
Biochemistry 27: 421-425.
[305] Rai, R. & Prasad, V. 1983. Studies on compatibility of nitrogen fixation by high tem‐
perature-adapted Rhizobium strains and Vigna radiata genotype at two levels in calca‐
reous soil. Journal of Agricultural Science 101: 377-381.
[306] Ramos, J. L., Galledgos, M. T., Marquéz, S., Ramos-Gonzalez, M. I., Espinosa Uergel,
M. & Segura, A. 2001. Responses of Gram-negative bacteria to certain environmental
stressors. Current Opinions in Microbiology 4: 166-171.
[307] Rao, A. V. & Venkateswarlu, B. 1983. Microbial ecology of the soils of Indian desert.
Agriculture, Ecosystems and Environment 10: 361-369.
[308] Ratnayake, M., Leonard, R. T. & Menge, J. A. 1978. Root exudation in relation to sup‐
ply of phosphorus and its possible relevance to mycorrhizal formation. New Phytol‐
ogist 81: 543-552.
[309] Redmond, J. W., Batley, M., Djordjevic, M. A., Innes, R. W., Kuempel, P. L. & Rolfe,
B. G. 1986. Flavones induce expression of nodulation genes in Rhizobium. Nature 323:
632-635.
[310] Reeve, W. G., Tiwari, R. P., Dilworth, M. & Glenn, A. R. 1993. Calcium affects the
growth and survival of Rhizobium meliloti. Soil Biology and Biochemistry 25: 581-586.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[311] Reeve, W. G., Tiwari, R. P., Wong, C. M., Dilworth, M. J. & Glenn, A. R. 1998. The
transcriptional regulator gene phrR in Sinorhizobium meliloti WSM419 is gulated by
Low pH and other stresses. Microbiology 144: 3335-3342.
[312] Rhoades, J. D., Chanduvi, F. & Lesch, M. 1999. Soil salinity assessment: Methods and
Electrical Conductivity Measurements. M5 ISBN 92-5-104281-0, FAO, Italy.
[313] Riccillo, P. M., Muglia, C. J., de Bruijn, F. J., Roe, A. J., Booth, I. R. & Aguilar, O. M.
2000. Glutathione is involved in environmental stress responses in Rhizobium tropici,
including acid tolerance. Journal of Bacteriology 182: 1748-1753.
[314] Rice, W. A., Olsen, P. E. & Collins, M. M. 1995. Symbiotic effectiveness of Rhizobium
meliloti at low temperature. Plant and Soil 170: 351-358.
[315] Richardson, A. E. & Simpson, R. J. 1989. Acid-tolerance and symbiotic effectiveness
of Rhizobium trifolii associated with a Trifolium subterraneum L. based pasture growing
in an acid soil. Soil Biology and Biochemistry 21: 87-95.
[316] Richardson, A. E., Djordjevic, M. A., Rolfe, B. G. & Simpson, R. J. 1988a. Effects of
pH, Ca and Al on the exudation from clover seedlings of compounds that induce the
expression of nodulation genes in Rhizobium trifolii. Plant and Soil 109: 37-47.
[317] Richardson, A. E., Simpson, R. J., Djordjevic, M. A. & Rolfe, B. G. 1988b. Expression
of nodulation genes in Rhizobium leguminosarum biovar trifolii is affected by low pH
and by Ca and Al ions. Applied and Environmental Microbiology 54: 2541-2598.
[318] Richardson, A. E., Djordjevic, M. A., Rolfe, B. G. & Simpson, R. J. 1989. Expression of
nodulation genes in Rhizobium and acid-sensitivity of nodule formation. Australian
Journal of Plant Physiology 16 (1): 117-129.
[319] Robertson, J. G., Lyttleton, P., Bullivant, S. & Grayston, G. F. 1978. Membranes in Lu‐
pin root nodules. I. The role of golgi bodies in the biogenesis of infection threads and
peribacteroid membranes. Journal of Cell Science 30: 129-149.
[320] Rodrigues, C., Laranjo, M. & Oliveira, S. 2006. Effect of heat and pH stress in the
growth of chickpea mesorhizobia. Current Microbiology 53: 1-7.
[321] Rokitko, P. V., Romanovskaia, V. A., Malashenko, L. R., Chernaia, N. A., Gushcha, N.
I. & Mikheev, A. N. 2003. Soil drying as a model for the action of stress factors on
natural bacterial populations. Microbiology 72: 756-761.
[322] Roth, L. E. & Stacey, G. 1989a. Bacterium release into host cells of nitrogen-fixing soy‐
bean nodules: the symbiosome membrane comes from three sources. European Jour‐
nal of Cell Biology 49: 13-23.
[323] Roth, L. E. & Stacey, G. 1989b. Cytoplasmic membrane systems involved in bacteri‐
um release into soybean nodule cells as studied with two Bradyrhizobium japonicum
mutant strains. European Journal of Cell Biology 49: 24-32.
185
186
Advances in Biology and Ecology of Nitrogen Fixation
[324] Rother, J. A., Millbank, J. W. & Thornton, I. 1983. Nitrogen fixation by white clover
(Trifolium repens) in grasslands on soils contaminated with cadmium, lead and zinc.
Journal of Soil Science 34: 127-136.
[325] Rup, L. 1988. Pesticide and nitrogen cycle. Volume III. CRC Press, Inc, Boca Raton,
Florida.
[326] Saeki, Y., Aimi, N., Tsukamoto, S., Yamakawa, T., Nagatomo, Y. & Akao, S. 2006. Di‐
versity and geographical distribution of indigenous soybean-nodulating bradyrhizo‐
bia in Japan. Soil Science and Plant Nutrition 52: 418-426.
[327] Saeki, Y., Ozumi, S., Yamamoto, A., Umehara, Y., Hayashi, M. & Sigua G. C. 2010.
Changes in population occupancy of bradyrhizobia under different temperature re‐
gimes. Microbes and Environments 25: 309-312.
[328] Sakamoto, K., Ogiwara, N. & Kaji, T. 2013. Involvement of autoregulation in the in‐
teraction between rhizobial nodulation and AM fungal colonization in soybean roots.
Biology and Fertility of Soils DOI 10.1007/s00374-013-0804-8.
[329] Salema, M. P., Parker, C. A., Kirby, D. K., Chatel, D. L. & Armitage, T. M. 1981. Rup‐
ture of nodule bacteria on drying and rehydration. Soil Biology and Biochemistry 14:
15-22.
[330] Sarkar, A., Nagarajan, R., Chaphadkar, S., Pal, S. & Singbal, S. Y. S. 1997. Contamina‐
tion of oraganochlorine pesticides in sediments from the Arabian Sea along the west
coast of India, Water Research 31: 195-200.
[331] Sarnaik, S. S., Kanekar, P. P., Raut, V. M., Taware, S. P., Chavan, K. S. & Bhadbhade,
B. J. 2006. Effect of application of different pesticides to soybean on the soil micro‐
flora. Journal of Environmental Biology 27: 423-426.
[332] Sauvage, D., Hamelin, J. & Larher, F. 1983. Glycine betaine and other structurally re‐
lated compounds improve the salt tolerance of Rhizobium meliloti. Plant Science Let‐
ters 31: 291-302.
[333] Sawicka, A. & Selwet, M. 1998. Effect of active ingredients on Rhizobium and Bradyr‐
hizobium legume dinitrogen fixation. Polish Journal of Environmental Studies 7 (5):
317-320.
[334] Saxena, N. P., Saxena, M. C., Johansen, C., Virmani, S. M. & Harris, H. 1996. Adapta‐
tion of chickpea in the west asia and north african region. ICRISAT, Hyderabad, In‐
dia; ICARDA, Aleppo, Syria.
[335] Schmeisser, C., Liesegang, H., Krysciak, D., Bakkou, N., Le Quere, A., Wollherr, A.,
Heinemeyer, I., Morgenstern, B., Pommerening-Roser, A., Flores, M., Palacios, R.,
Brenner, S., Gottschalk, G., Schmitz, R. A., Broughton, W. J., Perret, X., Strittmatter,
A. W. & Streit, W. R. 2009. Rhizobium sp. strain NGR234 possesses a remarkable num‐
ber of secretion systems. Applied and Environmental Microbiology 75: 4035-4045.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[336] Schoot Uiterkamp, A. J. M. 1990. Nitrogen cycling and human intervention, In
Gresshoff, P. M., Roth, L. E., Stacey G. & Newton W. E. (ed.), Nitrogen fixation: ach‐
ievements and objectives. Chapman and Hall, New York, N. Y., p. 55-66.
[337] Schultze, M. & Kondorosi, A. 2008. Regulation of symbiotic root nodule develop‐
ment. Annual Review of Genetics 32: 33-57.
[338] Sen, D., Baldani, J. I. & Weaver, R. W. 1990. Expression of heat induced proteins in
wild type and plasmidcured derivatives of Rhizobium leguminosarum by. trifolii. In:
Gresshoff, P. M., Roth, L. E., Stacey, G., Newton, W. E. (ed.): Nitrogen Fixation Ach‐
ievements and Objectives. Chapman and Hall, London, p. 200-201.
[339] Sepehri, M., Rastin, N. S., Rahmani, H. A. & Alikhani, H. 2006. Effects of soil pollu‐
tion by cadmium on nodulation and nitrogen fixation ability of native strains of Sino‐
rhizobium meliloti. Journal of Science and Technology of Agriculture and Natural
Resources 10: 153-163.
[340] Serraj, R. & Drevon, J. J. 1998. Effects of salinity and nitrogen source on growth and
nitrogen fixation in alfalfa. Journal of Plant Nutrition 21: 1805-1818.
[341] Serraj, R., Sinclair, T. R. & Purcell, L. C. 1999. Symbiotic nitrogen fixation response to
drought. Journal of Experimental Botany 50: 143-155.
[342] Shamseldin, A. & Werner, D. 2004. Selection of competitive strains of Rhizobium nod‐
ulating Phaseolus vulgaris and adapted to environmental conditions in Egypt, using
the gus-reporter gene technique. World Journal of Microbiology and Biotechnology
20: 377-382.
[343] Shime-Hattori, A., Iida, T., Arita, K., Park, K. S., Kodama, T. & Honda, T. 2006. Two
type IV pili of Vibrio parahaemolyticus play different roles in biofilm formation. FEMS
Microbiology Letters 264: 89-97.
[344] Shirkey, B., Kovarcik, D. P., Wright, D. J., Wilmoth, G., Prickett, T. F., Helm, R. F.,
Gregory, E. M. & Potts, M. 2000. Active Fe-containing superoxide dismutase and
abundant SodF mRNA in Nostoc commune (cyanobacteria) after years of desiccation.
Journal of Bacteriology 182: 189-197.
[345] Silva, K., Florentino, L. A., Silva, K. B., Brandt, E., Vandammme, P., Moreira, F. M. S.
2012. Cupriavidus necator isolates are able to fix nitrogen in symbiosis with different
legume species. Systematic and Applied Microbiology 35: 175-182.
[346] Singh, G. & Wright, D. 2002. In vitro studies on the effects of herbicides on the
growth of rhizobia. Letters in Applied Microbiology 35: 12-16.
[347] Singh, C. S., Lakshmi-Kumari, M., Biswas, A. & Subba Rao, N. S. 1973. Effect of car‐
bonate and bicarbonate of sodium on growth of rhizobia and nodulation in lucerne
(Medicago sativa L). Indian Journal of Microbiology 13: 125-128.
187
188
Advances in Biology and Ecology of Nitrogen Fixation
[348] Singleton, P.W. & Bohlool, B. B. 1984. Effect of salinity on nodule formation by soy‐
bean. Plant Physiology 74: 72-76.
[349] Smith, L. T. & Smith, G. M. 1989. An osmoregulated dipeptide in stressed Rhizobium
meliloti. Journal of Bacteriology 171: 4714-4717.
[350] Smith, L. T., Smith, G. M. & Madkour, M. A. 1990. Osmoregulation in Agrobacterium
tumefaciens: accumulation of a novel disaccharide is controlled by osmotic strength
and glycine betaine. Journal of Bacteriology 172: 6849-6955.
[351] Smith, L. T., Pocard, J. A., Bernard, T. & Le Rudulier, D. 1988. Osmotic control of gly‐
cine betaine biosynthesis and degradation in Rhizobium meliloti. Journal of Bacteriolo‐
gy 170: 3142-3149.
[352] Smith, L. T., Smith, G. M., Dsouza, M. R., Pocard, J. A., Le Rudulier, D. & Madkour,
M. A. 1994. Osmoregulation in Rhizobium meliloti: mechanism and control by other
environmental signals. Journal of Experimental Zoology 268: 162-165.
[353] Soria, R. S., Correa, N. S. & Rosas, S. B. 1996. Effect of water stress on the Guar-Bra‐
dyrhizobium system using PEG 6000. Phyton-International Journal of Experimental
Botany 58: 97-106.
[354] Soussi, M., Ocaña, A. & Lluch, C. 1998. Effects of salt stress on growth, photosynthe‐
sis and nitrogen fixation in chickpea (Cicer arietinum L.). Journal of Experimental Bot‐
any 49: 1329-1337.
[355] Soussi, M., Santamaria, M., Ocaña, A. & Lluch, C. 2001. Effect of salinity on protein
and lipoplysaccharide pattern in a salt-tolerant strain of Mesorhizobium ciceri. Journal
of Applied Microbiology 90: 476-481.
[356] Spaink, H. P. 2000. Root nodulation and infection factors produced by rhizobial bac‐
teria. Annual Review of Microbiology 54: 257-288.
[357] Sprent, J. I. & Sprent P. 1990. Nitrogen fixing organisms. Pure and Applied Aspects.
Chapman and Hall, Cambridge, p. 256.
[358] Staddon, W. J., Trevors, J. T., Duchesne, L. C. & Colombo, C. A. 1998. Soil microbial
diversity and community structure across a climatic gradient in Western Canada. Bi‐
odiversity and Conservation 7: 1081-1092.
[359] Stead, D. & Park, S. F. 2000. Roles of Fe superoxide dismutase and catalase in resist‐
ance of Campylobacter coli to freeze-thaw stress. Applied and Environmental Microbi‐
ology 66: 3110-3112.
[360] Steinborn, J. & Roughley, R. J. 1975. Toxicity of sodium and chloride ions to Rhizobi‐
um spp. in broth and peat culture. Journal of Applied Bacteriology 39: 133-138.
[361] Streeter, J. G. 1985. Accumulation of alpha, alpha-trehalose by Rhizobium bacteria and
bacteroids. Journal of Bacteriology 164: 78-84.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[362] Streeter, J. G. 2003. Effect of trehalose on survival of Bradyrhizobium japonicum during
desiccation. Journal of Applied Microbiology 95: 484-491.
[363] Streeter, J. G. & Bhagwat, A. 1999. Biosynthesis of trehalose from maltooligosacchar‐
ides in rhizobia. Canadian Journal of Microbiology 45: 716-721.
[364] Streeter, J. G. & Gomez, M. L. 2006. Three enzymes for trehalose synthesis in Bradyr‐
hizobium cultured bacteria and in bacteroids from soybean nodules. Applied and En‐
vironmental Microbiology 72: 4250-4255.
[365] Talibart, R., Jebbar, M., Gouesbet, G., Himdi-Kabbab, S., Wróblewski, H., Blanco, C.
& Bernard, T. 1994. Osmoadaptation in rhizobia: Ectoine-induced salt tolerance. Jour‐
nal of Bacteriology 176: 5210-5217.
[366] Talibart, R., Jebbar, M., Gouffi, K., Pichereau, V., Gouesbet, G., Blanco, C., Bernard, T.
& Pocard, J. A. 1997. Transient accumulation of glycine betaine and dynamics of en‐
dogenous osmolytes in salt-stressed cultures of Sinorhizobium meliloti. Applied and
Environmental Microbiology 63: 4657-4663.
[367] Tanabe, S., Iwata, H. & Tatsukawa, R. 1994. Global contamination by persistent orga‐
nochlorine and their ecotoxiological impact on marine mammals. Science of the Total
Environment 154: 163-177.
[368] Tester, M. & Davenport, R. 2003. Na+ tolerance and Na+ transport in higher plants.
Annals of Botany 91: 503-27.
[369] Thornton, F. C. & Davey, C. B. 1983. Acid dolerance of Rhizobium trifolii in culture
media. Soil Science Society of America Journal 47: 496-501.
[370] Tindall, B. J. 2008. The genus name Sinorhizobium Chen et al. 1988 is a later synonym
of Ensifer Casida 1982 and is not conserved over the latter genus name, and the spe‐
cies name 'Sinorhizobium adhaerens' is not validly published. Opinion 84 - Judicial
Commission of the International Committee on Systematics of Prokaryotes. Interna‐
tional Journal of Systematic and Evolutionary Microbiology 58: 1973-1973.
[371] Tiwari, R. P., Reeve, W. G., Dilworth, M. J. & Glenn, A. R. 1996a. An essential role for
actA in acid tolerance of Rhizobium meliloti. Microbiology 142: 601-610.
[372] Tiwari, R. P., Reeve, W. G., Dilworth, M. J. & Glenn, A. R. 1996b. Acid tolerance in
Rhizobium meliloti strain WSM419 involves a two-component sensor-regulator sys‐
tem. Microbiology 142: 1693-1704.
[373] Tong, Z. & Sadowsky, M. J. 1994. A selective medium for the isolation and quantifi‐
cation of Bradyrhizobium japonicum and Bradyrhizobium elkanii strains from soils and
inoculants. Applied and Environmental Microbiology 60: 581-586.
[374] Truchet, G., Roche, P., Lerouge, P., Vasse, J., Caumt, S., de Billy, F., Prome, J. & De‐
narie, J. 1991. Sulphated lipo-oligosaccharide signals of Rhizobium meliloti elicit root
nodule organogenesis in alfalfa. Nature 351: 670-673.
189
190
Advances in Biology and Ecology of Nitrogen Fixation
[375] Tu, J. C. 1981. Effect of salinity on Rhizobium- root hair interaction, nodulation and
growth of soyabean. Canadian Journal of Plant Science 61 (2): 231-239.
[376] Valverde, A., Velázquez, E., Gutiérrez, C., Cervantes, E., Ventosa A. & Igual, J. M.
2003. Herbaspirillum lusitanum sp. nov., a novel nitrogen-fixing bacterium associated
with root nodules of Phaseolus vulgaris. International Journal of Systematic and Evo‐
lutionary Microbiology 53: 1979-1983.
[377] Vance, C. P., Heichel, G. H. & Phillips, D. A. 1988. Nodulation and symbiotic nitro‐
gen fixation. In: Hanson, A. A., Barnes D. K. and Hill, R. R. (Eds), Alfalfa and Alfalfa
Improvement. Madison: American Society of Agronomy, p. 229-257.
[378] Vanderlinde, E. M., Harrison, J. J., Muszyński, A., Carlson, R. W., Turner, R. J. &
Yost, C. K. 2010. Identification of a novel ABC transporter required for desiccation
tolerance, and biofilm formation in Rhizobium leguminosarum bv. viciae 3841. FEMS
Microbiology Ecology 71 (3): 327-340.
[379] Vaneechoutte, M., Kampfer, P., De Baere, T., Falsen, E. & Verschraegen, G. 2004.
Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage includ‐
ing Ralstonia eutropha and related species, and proposal of Ralstonia (Pseudomonas) sy‐
zygii (Roberts et al. 1990) comb. Nov. International Journal of Systematic and
Evolutionary Microbiology 54: 317-327.
[380] Vincent, J. M., Thompson, J. A. & Donovan, K. O. 1961. Death of root nodule bacteria
on drying. Australian Journal of Agricultural Research 13: 258-270.
[381] Vinuesa, P., Silva, C., Lorite, M. J., Izaguirre-Mayoral, M. L., Bedmar, E. J. & Martí‐
nez-Romero, E. 2005. Molecular systematics of rhizobia based on maximum likeli‐
hood and Bayesian phylogenies inferred from rrs, atpD, recA and nifH sequences,
and their use in the classification of Sesbania microsymbionts from Venezuelan wet‐
lands. Systematic and Applied Microbiology 28: 702-716.
[382] Vriezen, J. A. C., de Bruijn, F. J. & Nüsslein, K. 2007. Responses of rhizobia to desicca‐
tion in relation to osmotic stress, oxygen and temperature. Applied and Environmen‐
tal Microbiology 73 (11): 3451-3459.
[383] Vriezen, J. A. C., de Bruijn, F. J. & Nusslein, K. 2006. Desiccation responses and sur‐
vival of Sinorhizobium meliloti USDA 1021 in relation to growth-phase, temperature,
chloride and sulfate availability. Letters in Applied Microbiology 42: 172-178.
[384] Wang, P., Zhong, Z., Zhou, J., Cai, T. & Zhu, J. 2008. Exopolysaccharide biosynthesis
is importantfor Mesorhizobium tianshanense: plant host interaction. Archives of Micro‐
biology 189: 525-530.
[385] Wang, X., Pan, Q., Chen, F., Yan, X. & Liao, H. 2011. Effects of coinoculation with ar‐
buscular mycorrhizal fungi and rhizobia on soybean growth as related to root archi‐
tecture and availability of N and P. Mycorrhiza 21: 173-181.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[386] Wani, P. A., Khan, M. S. & Zaidi, A. 2007a. Effect of metal tolerant plant growth pro‐
moting Bradyrhizobium sp. (vigna) on growth, symbiosis, seed yield and metal uptake
by green gram plants. Chemosphere 70: 36-45.
[387] Wani, P. A., Khan, M. S. & Zaidi, A. 2007b. Impact of heavy metal toxicity on plant
growth, symbiosis, seed yield and nitrogen and metal uptake in chickpea. Australian
Journal of Experimental Agriculture 47: 712-720.
[388] Wani, P. A., Khan, M. S. & Zaidi, A. 2008a. Effect of metal-tolerant plant growth-pro‐
moting Rhizobium on the performance of pea grown in metal-amended soil. Archives
of Environmental Contamination and Toxicology 55: 33-42.
[389] Wani, P. A., Khan, M. S. & Zaidi, A. 2008b. Impact of zinc-tolerant plant growth pro‐
moting rhizobacteria on lentil grown in zinc-amended soil. Agronomy for Sustaina‐
ble Development 28: 449-455.
[390] Weaver, R. W. & Holt, E. C. 1990. Short-term survival of rhizobia on arrow leaf clo‐
ver seed at different depths. Plant and Soil 122: 147-150.
[391] Weaver, R.W., Materon, L.A., Krautmann, M.E. & Rouquette, F.M. 1985. Survival of
Rhizobium trifolii in soil following inoculation of arrow leaf clover. MIRCEN Journal
of Applied Microbiology and Biotechnology 1: 311-318.
[392] Weisz, P. R., Denison, R. F. & Sinclair, T. R. 1985. Response to drought stress of nitro‐
gen fixation (acetylene reduction) rates by field-grown soybean. Plant Physiology 78:
525-530.
[393] Welsh, D. T. 2000. Ecological significance of compatible solute accumulation by mi‐
cro-organisms: from single cells to global climate. FEMS Microbiology Reviews 24:
263-290.
[394] Werner, M., Uehlein, N., Proksch, P. & Kaldenhoff, R. 2001, Characterization of two
tomato aquaporins and expression during the incompatible interaction of tomato
with the plant parasite Cuscuta reflexa. Planta, 213: 550-555.
[395] Wetzel, A. & Werner, D. 1995. Ecotoxicological evaluation of contaminated soil using
the legume root nodule symbiosis as effect parameter. Environmental Toxicology
and Water Quality 10: 127-134.
[396] Whelan, A. M. & Alexander, M. 1986. Effects of low pH and high Al, Mn, and Fe, lev‐
els on the survival of Rhizobium trifolii and the nodulation of subterranean colver.
Plant and Soil 92: 363-371.
[397] Whiteley, M., Bangera, M. G., Bumgarner, R. E., Parsek, M. R., Teitzel, G. M., Lory, S.
& Greenberg, E. P. 2001. Gene expression in Pseudomonas aeruginosa biofilms. Nature
413: 860-864.
191
192
Advances in Biology and Ecology of Nitrogen Fixation
[398] Wood, M. & Cooper, J. E. 1988. Acidity, aluminium and multiplication of Rhizobium
trifolii possible mechanisms of aluminium toxicity. Soil Biology and Biochemistry 20:
95-99.
[399] Wood, M., Cooper, J. E. & Bjourson, A. J. 1988. Response of Lotus rhizobia to acidity
and aluminium in liquid culture and in soil. Plant and Soil 107: 227-231.
[400] Xi, C., Schoeters, E., van der Leyden, J. & Michels, J. 2000. Symbiosis-specific expres‐
sion of Rhizobium etli casA encoding a secreted calmodulin-related protein. Proceed‐
ings of the National Academy of Sciences of the United States of America 97:
11114-11119.
[401] Yakoleva, Z. M. 1984. The effect of metal oxides on synthesis between Rhizobium trifo‐
lii and clover plants. Microbiologia 53: 308-312.
[402] Yao, P. Y. & Vincent, J. M. 1969. Host specificity in the root hair "curling factor" of
Rhizobium sp. Australian Journal of Biological Sciences 22: 413-423.
[403] Yildiz, F. H. & Schoolnik, G. K. 1999. Vibrio cholerae O1 El Tor, identification of a gene
cluster required for the rugose colony type, exopolysaccharide production, chlorine
resistance, and biofilm formation. Proceedings of the National Academy of Sciences
of the United States of America 96: 4028-4033.
[404] Young, J. M., Kuykendall, L. D., Martinez-Romero, E., Kerr, A. & Sawada, H. 2001. A
revision of Rhizobium Frank 1889, with an emended description of the genus, and the
inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de La‐
judie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R.
undicola and R. vitis. International Journal of Systematic and Evolutionary Microbiol‐
ogy 51: 89-103.
[405] Young, J. P., Crossman, L., Johnston, A., Thomson, N., Ghazoui, Z., Hull, K., Wexler,
M., Curson, A., Todd, J., Poole, P., Mauchline, T., East, A., Quail, M., Churcher, C.,
Arrowsmith, C., Cherevach, I., Chillingworth, T., Clarke, K., Cronin, A., Davis, P.,
Fraser, A., Hance, Z., Hauser, H., Jagels, K., Moule, S., Mungall, K., Norbertczak, H.,
Rabbinowitsch, E., Sanders, M., Simmonds, M., Whitehead, S. & Parkhill, J. 2006. The
genome of Rhizobium leguminosarum has recognizable core and accessory compo‐
nents. Genome Biology 7: 34. doi:10.1186/gb-2006-7-4-r34.
[406] Younis, M. 2007. Responses of Lablab purpureus-Rhizobium symbiosis to heavy metals
in pot and field experiments. World Journal of Agricultural Sciences 3(1): 111-122.
[407] Yousef, A. N. & Sprent, J. I. 1983. Effects of NaCl on growth, nitrogen incorporation
and chemical composition of inoculated and NH4NO3 fertilized Vicia faba (L.) plants.
Journal of Experimental Botany 34: 941-950.
[408] Yura, T., Kanemori, M. & Morita, M. T. 2000. The Heat shock response: regulation
and function: In Storz G. and Hengge-Aronis R. (ed.), Bacterial stress response, ASM
Press, Washington, D.C., p. 3-18.
Impact of Harsh Environmental Conditions on Nodule Formation and Dinitrogen Fixation of Legumes
http://dx.doi.org/10.5772/56997
[409] Zablotowicz, R. M. & Reddy, K. N. 2004. Impact of glyphosate on the Bradyrhizobium
japonicum symbiosis with glyphosate-resistant transgenic soybean: A minireview.
Journal of Environmental Quality 33 (3): 825-831.
[410] Zablotowicz, R. M., Eskew, D. L. & Focht, D. D. 1978. Denitrification in Rhizobium.
Canadian Journal of Microbiology 24: 757-776.
[411] Zahran, H. H. 1999. Rhizobium-legume symbiosis and nitrogen fixation under severe
conditions and in an arid climate. Microbiology and Molecular Biology Reviews 63:
968-989.
[412] Zahran, H. H. & Sprent, J. I. 1986. Effects of sodium chloride and polyethylene glycol
on root hair infection and nodulation of Vicia faba L. plants by Rhizobium leguminosa‐
rum. Planta 167: 303-309.
[413] Zahran, H. H., Räsänen, L. A., Karsisto, M. & Lindström, K. 1994. Alteration of lipo‐
polysaccharide and protein profiles in SDS-PAGE of rhizobia by osmotic and heat
stress. World Journal of Microbiology and Biotechnology. 10: 100-105.
[414] Zawoznik, M. S. & Tomaro, M. L. 2005. Effect of chlorimuron–ethyl on Bradyrhizobi‐
um japonicum and its symbiosis with soybean. Pest Management Science 61 (10):
1003-1008.
[415] Zhang, F., Smith, D. L. 1994. Effects of low root zone temperature on the early stages
of symbiosis establishment between soybean (Glycine max L. Merr) and Bradyrhizobi‐
um japonicum. Journal of Experimental Botany 45: 1467-1473.
[416] Zhang, F., Smith, D. L. 1995. Preincubation of Bradyrhizobium japonicum with genis‐
tein accelerates nodule development of soybean at suboptimal root zone tempera‐
tures. Plant Physiology 108: 961-968.
[417] Zhang, F. & Smith, D. L. 1996. Genistein accumulation in soybean (Glycine max L
Merr) root systems under suboptimal root zone temperatures. Journal of Experimen‐
tal Botany 47: 785-792.
[418] Zhang, F. & Smith, D. L. 1997. Application of genistein to inocula and soil to over‐
come low spring soil temperature inhibition of soybean nodulation and nitrogen fix‐
ation. Plant and Soil 192: 141-151.
[419] Zhang, F. & Smith, D. L. 2002. Interorganismal signaling in suboptimum environ‐
ments: The legume rhizobia symbiosis. Advances in Agronomy 76: 125-157.
[420] Zhang, J., Hamill, A. S. & Weaver, S. E. 1995. Antagonism and synergism between
herbicides: trends from previous studies. Weed Technology 9: 86-90.
193